bluuebunny/crossref_metadata_2025 huggingface dataset:
T cell-mediated bone marrow and splenic hypoplasia in a mouse model of acute graft vs. host disease
Authors: Enriquez Josue , Mims Brianyell McDaniel , Furr Kathryn , Grisham Matthew
Published: January 2023

A major limitation with use of hematopoietic stem cell transplantation to treat relapsing hematological malignancies is the development of a potentially lethal, multi-organ inflammatory disorder called acute graft versus host disease (aGVHD). Acute GVHD-associated bone marrow (BM) suppression and lymphoid tissue (LT) hypoplasia creates protracted immunodeficiency that greatly increases the risk of infections, bleeding and death.

Objective

We wished to define the role that allogeneic CD4+ T cells play in a mouse model of aGVHD-associated BM and LT aplasia that does not require lethal myeloablative conditioning.

Methods

Allogeneic CD4+CD25−T cells (0.5×106 cells) obtained from Bl6-H2-Ab1bm12 (Bl6-BM12) donor mice were adoptively transferred into sub-lethally irradiated C57Bl6 (Bl6) recipients. Mice were monitored daily for clinical signs of aGVHD.

Results

Adoptive transfer of allogeneic but not syngeneic T cells induced a time-dependent loss of survival and remarkable reductions of cellularity in the BM and spleen. Virtually all allogeneic mice developed severe anemia at 15 days post transfer. Flow cytometric analyses revealed dramatic and significant losses of CD4+ T cells, myeloid cells and NK cells in the BM and spleen indicating BM suppression and spleen hypoplasia. Interestingly, we observed little immune cell infiltration in the lungs, liver and skin of allogeneic recipients and no evidence of disease in any tissue of syngeneic mice. These data suggest that the major target tissues in this model of aGVHD are BM and spleen.

Conclusions

When taken together, these data demonstrate that adoptive transfer of allogeneic CD4+ T cells into sub-lethally irradiated recipients induces aGVHD-associated immunodeficiency.
IL-33 signaling in the barrier tissues sustains alloimmune responses mediating graft vs. host disease
Authors: Dwyer Gaelen K , Mathews Lisa , Lucas Anna , Blazar Bruce R. , Turnquist Heth R
Published: January 2023

Objective

Graft vs. host disease (GVHD), where donor T cells recognize and destroy alloantigen-expressing host tissues, is a major complication after allogeneic bone marrow transplantation (alloBMT). To create space for donor stem cells and remove recipient immune cells, alloBMT protocols rely on conditioning regimens involving irradiation and/or chemotherapy. Interleukin (IL)-33 is augmented in recipient barrier tissues by conditioning before alloBMT and is a required signal to donor T cells for GVHD. In the current studies, we tested the hypothesis that the dominant role for IL-33 in GVHD lethality is to act as a sustaining signal to allogeneic T helper Type 1 (Th1) effectors in the barrier tissues.

Methods

To establish the role of IL-33 in CD4+ T cell effector function in the target tissue during GVHD, we used two CD4+-driven GVHD models. In the first, irradiated Il33−/− and Il33+/+ B6 recipients were given BALB/c alloBMT and T cells. In the second, irradiated Il33−/− and Il33+/+ bm12 recipients were given BMT and CD90.1+ B6 T cells. Donor T cell responses were characterized by flow cytometry at day 7, 14, and 28 post alloBMT.

Results

B6 Il33−/− recipients had significantly reduced frequency of IL-33 receptor-expressing CD4+Tbet+ cells in the small intestine lamina propria (SI LP), but not in the spleen (SPN) at day 7. In the MHCII-disparate B6 to bm12 GVHD model, IL-33 deficient recipients had profoundly reduced overall CD4+ donor T cells in the SI LP, but not in the SPN or lymph nodes (LN) at both day 14 and 28.

Conclusions

Our data reveal that IL-33 is not critical for donor Th1 responses in the SPN and LN, but necessary for Th1 cells to persist in barrier tissues such as the SI LP after alloBMT.
The effect of mouse cytomegalovirus infection on natural killer cell development following hematopoietic stem cell transplant
Authors: Dunai Cordelia , Aguilar Ethan G. , Khuat Lam T. , Le Catherine T. , Wang Ziming , Stoffel Kevin M. , Murphy William J
Published: January 2023

Hematopoietic stem cell transplant (HSCT) is a treatment for patients with hematological malignancies who are not eligible to receive intensive cytoreductive therapy. Cytomegalovirus (CMV) is an extremely prevalent infection and in immune-compromised patients, it is a significant cause of morbidity and mortality. Approximately two-thirds of seropositive patients experience CMV reactivation following HSCT. There have been reports that CMV reactivation causes increased activation of NK cells which actually benefits graft-versus-tumor effects. Our study objective is to delineate the kinetics of this effect and determine whether there are long-term functional differences in NK cells. We hypothesized that immune reconstitution is impacted by CMV infection and that the lymphopenic and inflammatory environment post-HSCT detrimentally affects the immune response to CMV infection. Using a syngeneic HSCT model in C57BL/6 mice, we studied de novo NK cell repopulation. At day 8 post-transplant, mice were inoculated with a low dose of mouse CMV. We found a significantly higher viral burden in the HSCT recipients compared to control mice. We found that CMV-specific NK cells (Ly49H+) rapidly expanded following CMV infection post-HSCT, but experienced a population collapse after two weeks. This is possibly due to exposure to a primary virus infection early on in development and/or the increased viral burden. There was a higher frequency of mature NK cells and IFN-gamma producing NK cells following CMV infection in the HSCT environment, suggestive of increased activation and accelerated differentiation. The disproportionate loss of the Ly49H+ NK cells may cause long-term functional defects in the HSCT recipient immune response.
ITK signaling differentiates GVT and GVHD after allogeneic bone marrow transplantation by regulating IRF-4, JAK/STAT and Eomesodermin expression
Authors: Karimi Mobin , August Avery , Kambayashi Taku , Huang Weishan , Jordan Martha
Published: January 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for patients with hematologic malignancies. Donor T cells contained within the graft prevent tumor recurrence by exhibiting graft-versus-tumor (GVT) effects but also cause graft-versus-host disease (GVHD); therefore, novel treatment strategies are needed to maintain GVT while suppressing GVHD. We investigated the role of TCR-mediated ITK activation in mediating GVT vs. GVHD effects after allo-HSCT. We discovered that T cells from ITK−/−mice display a CD62Lhi CD122+ CD44hi Innate Lymphoid Cell-Like (ILCL) functional phenotype, and higher Eomes but not T bet expression compared to WT. We showed that T cells from ITK−/−mice reduced IRF-4, JAK1 and JAK2 and STAT3. T cells from ITK−/− mice displayed reduced cytokine production but showed preserved cytotoxicity after allo-HSCT. These cells also showed defective upregulation of the chemokine receptors CX3CR3, CXCR1, and CXCR6, which correlated with their reduced migration into GVHD target organs. The defective migration of ITK deficient T cells into GVHD target organs contributed to separation of GVHD and GVT effects, since ITK deficeint T cells cleared intravenously injected but not subcutaneously injected tumor cells in allo-HSCT mice. Moreover, pharmacological ITK inhibition attenuated GVHD and preserved GVT function by wild type CD8+ T cells. Together, our data suggest that ITK inhibition could be used as therapy after allo-HSCT to reduce GVHD while preserving the beneficial GVT effects by donor T cells
Vasoactive intestinal polypeptide synthesis by somatic tissues in transplant recipients limits GVHD in allo-BMT
Authors: Li Yiwen , Waller Edmund K
Published: January 2023

Both donor and recipient hematopoietic cells regulate GvHD in allo-BMT, but the role of somatic host tissues in the induction and maintenance of GvHD is less clear. Vasoactive Intestinal peptide (VIP) is a neuropeptide with potent immune-regulatory activity. Our goal was to examine the role of VIP synthesis by recipient tissues on donor T cell activation and GvHD in allo-BMT. We used MHC mis-matched allo-BMT models, B10.BR-->B6 and Balb/c-->B6, irradiating WT and VIP-KO B6 mice with 11Gy on day −1 and transplanting with 5 × 10E6 T cell depleted (TCD) bone marrow (BM) plus 0, 1 × 10E6, or 3 × 10E6 T cells from MHC mis-matched donors. B6-->B6 radiation chimeras lacking VIP in either hematopoietic or non-hematopoietic cells were created by performing B6 WT-->VIP-KO and VIP-KO-->WT syngeneic transplants. Production of VIP in transplant recipients was visualized with immune-fluorescent staining of tissues using an anti-VIP antibody. Transplanting TCD BM plus 3 × 10E6 donor T cells from B10.BR or Balb/c donors in VIP KIO mice led significantly increased GVHD-mortality compared with WT recipients. Donor-derived T cells in VIP-KO recipients had significantly higher Th1 and Th17 polarization. B10.BR-->B6 second allogeneic transplantation of radiation chimeras caused lethal GVHD mortality in VIP-KO-- >VIP-KO and WT-->VIP-KO mice, but not in WT-->WT or VIP-KO-->WT B6 mice, demonstrating the protective effect of VIP was due to synthesis by somatic recipient cells. Immunofluorescent imaging of allo-BMT recipients showed up-regulation of VIP in lungs post-transplant and high VIP production within neurons innervating the lungs. Short-term administration of VIP from D0 to D10 prevented GvHD-mortality in VIP-KO recipients transplanted BM & T cells.
Granzyme A-producing helper T cells are critical for lethal acute graft-versus-host disease
Authors: Olson Matthew Richard , Park Sungtae , Griesenauer Brad , Mehrpouya-Bahrami Pegah , Kazemian Majid , Imam Tanbeena , Srivastava Rajneesh , Hayes Tristan , Pardo Julian , Janga Sarath Chandra , Paczesny Sophie , Kaplan Mark H
Published: January 2023

Graft-versus-host disease (GVHD) is a deleterious allo-immune response that occurs in patients undergoing hematopoietic stem cell transplantation (HSCT) for treatment of blood-derived malignancies. CD4 T helper (Th) cells within the donor HSC pool recognize allogeneic HLA molecules and traffic to multiple organs, including the intestines where increased inflammation correlates with worsened clinical prognosis. Despite the known role of Th cells in GVHD, the means by which they promote disease remain unclear. In our studies, we have identified a novel class of Th cells that produce the serine protease granzyme A (GrA) within the intestines of mice succumbing to GVHD. GrA+ Th cells were distinct from other Th lineages and largely lacked co-expression of other Th lineage-associated cytokines or cytolytic mediators. Importantly, mice that received HSCT with GrA-deficient CD4 T cells exhibited significantly reduced morbidity and mortality as compared to controls, suggesting that Th-derived GrA is critical for GVHD progression. In vitro, differentiation of GrA+ Th cells was optimally induced with a combination of IL-6, IL-21 and IL-4 in a STAT3- and STAT6-dependent fashion. In vivo, STAT3- was also partially required for the differentiation of GrA+ Th cells during the GVHD response. In summary, GrA+ Th cells represent a novel STAT3-dependent Th cell type that is critical for allo-immune mediated disease.
Robust enhancement of tumor-dictated CD8 T cell trafficking and activation by QS21 adjuvanted vaccine
Authors: Sastry Jagannadha K , Nookala Sita M , Dorta-Estremera Stephanie M , Sierra Gloria , Yanamandra Ananta V.
Published: January 2023

Adjuvants enhance immune response to antigens and increase vaccine efficacy. We investigated the relative potency of adjuvants with diverse mechanism of action: a natural killer T cell activating ligand (alpha-galactosylcermide, αGalCer), a toll-like receptor 9 ligand (CpG B), a liposomal nanoparticle (R-DOTAP) and a plant saponin from Quillajira saponaria (QS21). We used OVA TCR transgenic model and also compared systemic vs mucosal routes of delivery for inducing antigen-specific CD8 T cell responses in naïve and syngeneic tumor-bearing mice. Wild type C57Bl/6 mice were vaccinated with OVA admixed with the adjuvants via subcutaneous (SC) or intranasal (IN) route 1d after adoptive transfer of the OT-I cells. Single cell suspensions of tissues 7d post-vaccination were analyzed using flow cytometry for OT-1 cells (CD45.1+Vα2+). Vaccination by either SC or IN route using QS21 adjuvant showed the most potent trafficking and activation of CD8+ T cells to both systemic and at mucosal sites, with the highest response observed in the lungs. Vaccination using the αGalCer by the IN route was relatively more effective than when delivered by the SC route, but the responses with αGalCer were lower compared to that with the QS21 adjuvant. However, vaccination of mice bearing B16F10-OVA resulted in preferential trafficking of OT-I cells to the tumors with much reduced numbers at the other tissues. Once again, these responses were robustly higher with QS21 vaccine delivered by either SC or IN route and the OT-1+ T cells from the tumors exhibited higher levels of functional and proliferating properties based on IFN-γ and Ki67 staining. Our results demonstrate that QS21 is a potent adjuvant for prophylactic as well as therapeutic vaccination applications.
Defining the signaling and downstream regulation of neutrophil-dendritic cell development for adjunctive therapy
Authors: Gui Michael , Fites J. Scott , Sykes David , Mansour Michael K , Klein Bruce S
Published: January 2023

Neutrophils are typically considered short-lived and terminally differentiated. However, recent studies have identified a poorly understood neutrophil subset that can differentiate into dendritic cells and become long-lasting “hybrids” of neutrophils and dendritic cells. These neutrophil-dendritic cells (PMN-DCs) maintain the functions of canonical neutrophils while gaining antigen-presenting properties of dendritic cells, enabling them to contribute to both innate and adaptive immunity. Although PMN-DCs have been characterized in various inflammatory conditions, the cues that drive their development are not fully understood. Granulocyte macrophage colony-stimulating factor (GM-CSF) has previously been implicated as an important signal for PMN-DC development in studies of murine and human neutrophils. In vivo neutralization suggests that GM-CSF is sufficient but not necessary to drive PMN-DC differentiation. To determine the requirement of GM-CSF we have deleted GM-CSF receptor using CRISPR editing of a murine neutrophil progenitor cell line. We have tested other implicated cytokines in vitro, including IL-3, IL-4, TNF-α and IFN-γ, however these signals alone do not induce differentiation of PMN-DCs but can augment or blunt PMN-DC differentiation. Previous work shows that PMN-DCs are potent effectors of antifungal immunity through both direct killing and antigen presentation; understanding of the developmental signals that drive PMN-DC differentiation is essential for any future therapies targeting PMN-DCs.
Monocytes outperform ex vivo generated dendritic cells as cellular vaccines to trigger cytotoxic T lymphocyte responses against cancer in pre-clinical models
Authors: Huang Min-Nung , Nicholson Lowell T. , Batich Kristen A. , Kopin David , Swartz Adam M. , Sampson John H , Gunn Michael Dee
Published: January 2023

Conventional dendritic cell (DC) vaccines using ex vivo generated DC have been widely tested in clinical trials in the treatment of human cancers. These DC vaccines have often displayed limited efficacy despite a complex manufacturing process. Although the efficacy of DC vaccines is generally thought to depend on their antigen-presenting cell function, several lines of evidence suggest that, instead of directly presenting antigen (Ag) to naïve T cells, administered DC trigger cytotoxic T lymphocyte (CTL) responses in vivo mainly by transferring Ag to lymphoid-resident conventional DC (cDC). We tested here whether monocytes, known to transfer Ag to cDC in vivo, can be directly loaded with tumor Ag to trigger robust anti-tumor CTL responses. We found that IV administered monocytes loaded with protein or peptide Ag triggered stronger Ag-specific CTL responses than conventional adjuvant-based and DC vaccines. In several murine cancer models, including intra-cranial glioblastoma, we showed that monocyte vaccination suppressed tumor growth better than conventional adjuvant-based, GVAX and DC vaccines. Such anti-tumor activities could be further enhanced with immune checkpoint inhibition. Ag-loaded monocytes did not directly activate naïve CD8+T cells. Instead, they formed stable physical contacts with and transferred Ag to splenic CD8+cDC. This Ag transfer required the formation of connexin 43-containing gap junctions between monocytes and CD8+cDC. Our findings demonstrated that gap junctions are an efficient Ag transfer pathway in vivo between monocytes and splenic CD8+cDC and suggested that IV administered Ag-loaded monocytes may serve as a simple and efficacious cellular vaccine platform for the treatment of human cancers.
Engineered Toxin Bodies delivering immunogenic MHC class I peptides to tumor cells summon polyfunctional and relevant CTL responses against cancers
Authors: Dekker Joseph D , Brieschke Brigitte , LeMar Sara , Cornelison Garrett L. , Iberg Aimee , Robinson Garrett L. , Chu Shaoyou , Willert Erin K , Ramos Hilario J.
Published: January 2023

Immunotherapeutic approaches such as CAR-T and T cell engaging bispecific antibodies have shown positive clinical outcomes and demonstrate the value of harnessing cytotoxic T lymphocyte (CTL) activity for tumor intervention. Here we describe Engineered Toxin Bodies (ETBs) with Antigen Seeding Technology (AST) as a unique tumor targeted therapeutic approach to recruit endogenous polyfunctional memory CTL responses against cancers.

Molecular Templates’ ETB platform combines antibody domains with a proprietary form of the Shiga like Toxin A (SLTA) subunit to specifically target cells and exploit SLTA’s intrinsic ability to self-internalize and destroy ribosomes. With the addition of a foreign class I antigenic peptide fused to an ETB (AST), antigens present in complex with endogenous MHC I to initiate a high avidity T cell response to target cells. Utilizing this approach, we demonstrate ETBs targeted to clinically relevant markers can seed antigenic peptides to hematological tumor, solid tumor, and primary cells.

Expanded CTLs from donors with preexisting Human Cytomegalovirus (CMV) reactivity phenotypically resembled memory expansion from natural infection. We next delivered class I CMV peptide to tumor cells by AST with ETBs targeted to PD-L1 and co-cultured tumor cells with expanded CTLs. This led to activation of CMV specific T cells, direct tumor-T cell engagement, and potent cell kill activity above that of parental ETBs. CTL phenotyping and cytokine profiling revealed polyfunctional T cell activation driven by AST enabled ETBs. Thus, ETBs delivering peptide via AST provide additive mechanisms of action: direct cell kill via ribosomal destruction, and antigen specific polyfunctional memory CTL activation.
Intradermal DNA Aβ42 immunization via needle-less Jet injection as potential immunotherapy for Alzheimer’s disease
Authors: Lambracht-Washington Doris , Fu Min , Rosenberg Roger N
Published: January 2023

The most common form of dementia found in the elderly is Alzheimer’s disease (AD) and AD progression has been strongly associated with accumulation of Amyloid beta 1–42 (Aβ42) and neurofibrillary tangles (tau) in brain. Amyloid pathology precedes tau pathology and an immune-mediated approach to reduce amyloid plaques without causing brain inflammation is highly desirable for possible clinical use. Genetic immunization, in which the immunizing agent is DNA encoding Aβ42, has great potential because the immune response to DNA delivered into the skin is generally non-inflammatory, and differs qualitatively from immune responses elicited by peptides, which are inflammatory with cellular production of IFNγ and IL-17. DNA immunization has been difficult in larger mammals and a potential barrier is the method for delivery of the DNA antigen. Over the last decade substantial progress has been made to increase the effectiveness of DNA delivery and immune responses, respectively. Recently, needless DNA Jet injection was approved by the FDA for a Zika vaccine. We are currently working on translating our established gene gun DNA immunization approach in mice into the clinically more feasible method of DNA delivery via jet injection. We have tested the Jet injector alone and in combination with electroporation in wild-type mice and New Zealand White rabbits as a large mammal. We found good antibody production and safe cellular immune responses (no inflammatory cytokines) in both species. Antibody levels and kinetics in mice and rabbits were very similar with about 30 μg of anti-Aβ antibodies after four immunizations. This study is designed to optimize DNA delivery for possible testing of the DNA Aβ42 vaccine for AD prevention in a future clinical trial.
Immunotherapy with tumor membrane vesicle-based cancer vaccine inhibits metastatic tumor growth in lung cancer model
Authors: Bommireddy Ramireddy , Huang Haley Lei , Munoz Luis E , Menon Ashwathi P , Ramalingam Suresh , Selvaraj Periasamy
Published: January 2023

Lung cancer is the leading cause of cancer related deaths accounting for approximately 30% of all cancer related mortalities affecting both men and women. Currently, there is no cure for the common non-small cell lung cancer (NSCLC) thus development of efficacious therapies is urgently needed. In this study, we investigate the efficacy of tumor membrane-based vaccine immunotherapy and immune checkpoint blockade antibodies lung cancer models LL/2 and CMT-167 models. LL/2 and CMT-167 tumor tissues were harvested from C57BL/6 mice and processed to generate tumor membrane vesicles (TMVs). TMVs were then modified with immunostimulatory molecules and used for immunization. Tumor growth was monitored every 3 days. Survival was assessed using a Kaplan-Meier survival curve and significance determined using a Log-rank test for comparison analysis. Metastasis of CMT-167 was assessed by clonogenic assay. TMV vaccine alone, or in combination, anti-PD-1 or anti-CTLA-4 antibodies neither decreased the tumor growth nor improved the survival of mice challenged with LL/2 tumor cells. The prophylactic vaccination of TMV vaccine prevents the CMT-167 tumor growth in mice and helps the mice develop immunological memory to the tumor cell challenge. In a therapeutic setting, the TMV vaccine inhibited tumor growth and induced primary tumor regression in 40% of mice and significantly reduced the metastatic tumor burden. Anti-tumor immunity induced by TMV vaccine requires CD4+ and NK cells albeit to a lesser extent but not CD8+ T cells for controlling the tumor growth. These models provide a valuable tool in analyzing the mechanism of interaction of immune cells with tumors that lead to either tumor regression or resistance to immunotherapies.
CD38hi B-regulatory (B-reg) cells maintain pathological immune tolerance in chronic lymphocytic leukemia (CLL)/B cell diseases: Potential therapeutic considerations
Authors: Manna Alak , Aulakh Sonikpreet , Sher Taimur , Ailawadhi Sikander , Chanan-Khan Asher , Paulus Aneel
Published: January 2023

Chronic Lymphocytic Leukemia (CLL) is considered a disease of antigen-naive CD5+CD23+CD19+κ/λ+ B-lymphocytes, in which the tumor microenvironment is highly immunosuppressive. Among several human B cell subsets, B10 [CD24hiCD27+CD38hi]/B regulatory cells (Bregs) are functionally discernable in patients with aggressive CLL as they support and regulate immunological tolerance via production of IL-10 and transforming growth factor β (TGF-β). In an ex vivo setting, we noted CD38hi CLL Bregs to promote transformation and expansion of CD4+CD25+FoxP3+Tregs; an effect that was abrogated in the presence of IL10/TGF-β neutralizing antibodies. In patients with CLL, Bregs also prohibit the expansion of cytotoxic T cells (CD8+T cells; Tc), natural killer (NK) cells and other pro-inflammatory lymphocytes (CD4+ helper T cells; Th1 and Th17). We noted that patients with CLL had a significantly higher % of Tregs (11.8±2.0) compared to healthy donors (1.9±0.3) and similar to Bregs, a substantial proportion of CLL Tregs had high CD38 expression (MFI=616.8±36.2). We demonstrated that an anti-CD38 therapeutic antibody was able to eliminate CD38+ Bregs and Tregs from CLL patients by immune effector mechanisms (ADCC, CDC, ADCP) as well as direct mitochondrial/FcγR-mediated apoptosis. Furthermore, in a PDX model of CLL, mice treated with anti-CD38 therapy had decreased % Bregs and Tregs but increased Th17 and CD8+ T-cells (vs. vehicle; p<0.05). These observations carry implications beyond CLL for any Breg/Treg-dependent cancer or disease (i.e. Systemic Lupus Erythematosus, Rheumatoid arthritis, Sjögren’s syndrome), wherein CD38-targeted therapy could potentially rescue/improve an immune tolerant microenvironment by eradication of Bregs.
A novel preclinical immunocompetent CAR T cell mouse model for solid tumors
Authors: Sullivan Peter M , Arguedas-Jimenez Laura , Johnson Adam , Yokoyama Jason , Yuzefpolskiy Yevgeniy , Jensen Michael , Kalia Vandana , Sarkar Surojit
Published: January 2023

Chimeric antigen receptor (CAR) T cells therapies have made substantial progress in treating blood cancers in humans. However, CAR T therapies currently have several limitations, including the lack of a persistent anti-tumor memory population to limit relapse and poor efficacy against solid tumors. Many studies on CAR T therapies have used immunocompromised xenograft mouse models, limiting the translational impact of the results. Furthermore, mouse models have largely focused on cancers in the blood while neglecting solid tumors. To improve the relevance of preclinical studies of CAR T cell therapy and to understand the mechanisms underlying some of the current CAR T therapy limitations, we have created a novel immunocompetent mouse model. This model consists of three components: murine CD8 T cells expressing the human CD19 CAR, syngeneic murine tumor cell lines expressing a truncated human CD19 (CD19t) serving as the tumor antigen, and an immunocompetent mouse tolerized to human CD19t and the CD19 CAR. Our results demonstrate that the CD19 CAR T cells can efficiently target the CD19t-expressing tumor lines. Furthermore, tolerized recipient mice do not reject implanted syngeneic CD19t-expressing tumors or adoptively transferred CD19 CAR-expressing T cells. This model now provides a platform to study the in vivo biology of CAR T cells with respect to localization, exhaustion, and functional reinvigoration; to investigate best approaches to treating multiple solid tumors with distinct microenvironments; and to test the preclinical efficacy of novel CAR T strategies.
Covalent adaptor synNotch and chimeric antigen receptors (CARs) for programmable antigen targeting
Authors: Lomueller Jason Jakob , Butchy Adam , Tivon Yaniv , Kvorjak Michael , Miskov-Zivanov Natasa , Deiters Alexander , Finn Olivera J
Published: January 2023

Chimeric antigen receptors (CARs) are artificial T cell receptors that re-target patients’ T cells to specifically bind and kill tumor cells. Adoptive cell therapy with CAR T cells targeting CD19 has revolutionized treatment of refractory B cell acute lymphoblastic leukemia, and there is great interest in generating CAR T cells treating other cancers by targeting additional tumor antigens. Another promising class of engineered receptors are synthetic Notch (synNotch) receptors that can sense an antigen of interest on a neighboring cell and turn on expression of any transgene(s) of interest. To expand the targeting capabilities of these receptors, we have developed “universal” CAR and SynNotch receptors whose antigen-specificity can be re-directed by co-administered tumor-specific antibodies. Instead of directly targeting a tumor antigen, our universal receptors contain the SNAPtag self-labeling enzyme, which reacts with antibodies conjugated to benzylguanine (BG) to post-translationally assemble complete covalently associated antigen receptors. We demonstrate that the activation of SNAP CAR and SNAP-SynNotch receptors can be successfully re-targeted by several clinically relevant antibodies including: Rituximab, FMC63, Herceptin, and Cetuximab. SNAP-SynNotch cells demonstrate potent transgene activation, and SNAP-CAR T cells are capable of performing effector functions in a BG-antibody-directed antigen-specific manner. Additionally, the receptor response is titratable by BG-antibody dose. Finally, a continuous mathematical model was constructed to describe and optimize system activity. SNAP synNotch and SNAP CAR T cells provide a powerful new strategy to retarget engineered cells to multiple antigens.
Improving immunotherapy through immune checkpoint assays, CAR-T target display, and effector function optimization
Authors: Edgcomb Stephen , Zhu Jie , Tao Zhihua , Hsu Matthew , Kimbara Michelle , Oberle Suzan , Zhu Henry
Published: January 2023

Cancer and autoimmune disease lead to illness by damaging and evading a patients natural defenses. Immunotherapy methods are designed to reclaim and bolster the immune system to treat disease. This requires both highly specific binding of therapeutic agents to target molecules and optimizing techniques to harness the effector functions of the immune system. We describe methods for both these requirements. These include cell based systems for improving attachment to targets by assaying binding of therapeutic antibodies to immune checkpoint receptors and displaying molecular targets to CAR- T cells. They also include assays to improve immune effector functions such as Antibody-dependent cell-mediated cytotoxicity (ADCC) through cell based assays of IgG Fc binding to FCgRs.
Novel HIV DNA vaccine changes the immune hierarchy and elicits broad cytotoxic T cell responses targeting subdominant viral epitopes
Authors: Felber Barbara K. , Lu Zhongyan , Hu Xintao , Valentin Antonio , Rosati Margherita , Mullins James I. , Pavlakis George N.
Published: January 2023

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting non-essential variable regions are hurdles in the development of an effective AIDS vaccine. We developed a DNA vaccine comprising conserved elements (CE) of Gag and Env. Macaques (RM) received the CE DNA prime/CE+full-length DNA co-delivery booster vaccine regimen via intramuscular injection followed by electroporation. Priming vaccination with CE DNA is critical to efficiently overcome the dominance imposed by Gag and Env variable regions. CE vaccine induced broad, potent and durable cytotoxic T cell responses targeting conserved segments. Gag CE-specific T cells showed robust anamnestic responses upon infection with SIVmac239 which led to the identification of CE-specific cytotoxic lymphocytes able to recognize several CE epitopes presented on SIV infected cells in vivo. The T cell responses induced by the HIV Env CE immunogen were also recalled upon SIV infection, leading to the identification of two cross-reactive epitopes between HIV and SIV Env. These data demonstrate that the CE DNA vaccines changed the normal immunodominance patterns, eliciting immune responses that included subdominant, highly conserved epitopes. These vaccine regimens augment cytotoxic T cell responses to highly conserved epitopes in the viral proteome and maximize response breadth. The HIV Gag CE vaccine regimen is evaluated for prevention and immunotherapy in clinical trials (HVTN119 and ACTG 5369).
Influence of vaccine-induced mucosal NKp44+ and mucosal NKp44−NKG2A− cells on SIVinfection outcomes in rhesus macaques
Authors: RAHMAN MOHAMMAD ARIF , Ko Eunju , Enyindah-Asonye Gospel , Hait Sabrina Helmold , Hoang Tanya , Robert-Guroff Marjorie
Published: January 2023

NK cells play an essential role in control of viral infection. We investigated NK cell dynamics and function post-vaccination with replicating adenovirus (Ad)-SIV recombinants and SIV envelope and subsequent intravaginal exposure to SIV. Macaques that became infected were studied. As no differences in NK cell frequencies or cytokine production were seen between vaccinated and Ad-empty/alum controls, suggesting responses observed were due to the Ad-vector and alum immunizations, the groups were combined. Mucosal NKp44+ NK cells increased post-vaccination and returned to pre-levels post-infection. Post-vaccination NKp44+ cell frequencies tended to correlate (p = 0.070, r = 0.683) while NKp44+IL-17+ cell frequencies positively correlated (p = 0.042, r = 0.742) with number of challenges for SIV acquisition. Additionally, negative correlations with peak viral load (VL) (p = 0.0072, r = −0.881) and chronic median VL (p = 0.0107, r = −0.857) suggested that these IL-17+ cells might inhibit SIV acquisition by promoting epithelial integrity and mucosal homeostasis. In contrast, the frequency of NKp44−NKG2A−double negative (DN) cells, a less-defined innate lymphoid cell population, decreased post-vaccination, but returned to pre-level post-infection. Post-vaccination DN cells (p = 0.042, r = −0.743) and IFN- γ+DN cells (p = 0.038, r = −0.755) negatively correlated with number of SIV challenges and positively correlated with chronic median VL (p = 0.046, r = 0.738) and peak VL (p = 0.001, r = 0.952), suggesting both cell populations might enhance SIV acquisition and contribute to viremia. No associations of circulating NK cells with protection were seen. Vaccines that promote mucosal NKp44+ cells and suppress DN cells are likely desirable.
Antibody cross-reactivity in chronic HIV/HCV co-infection
Authors: Pilewski Kelsey , Setliff Ian , Kalams Spyros , Georgiev Ivelin
Published: January 2023

HIV/HCV co-infected individuals fair worse than their mono-infected counterparts, experiencing rapid progression to fibrosis, and increased chance of liver damage due to HAART. Given this, novel antibody therapeutic and rational vaccine design for both viruses have become top priorities worldwide. This study seeks to understand the antibody response in chronic HIV/HCV co-infection. Normally selected against, antibodies that recognize diverse antigens confer a selective advantage against highly mutable pathogens, such as HIV and HCV. We hypothesized that chronic co-infection leads to the development of antibodies that can recognize both viruses.

In collaboration with the Vanderbilt HIV cohort, we obtained patient PBMCs from a single HIV/HCV co-infected African American man, at four timepoints between 0.5 – 3 years post HIV infection (ypi). For 0.5, and 1.5 ypi we performed antigen-specific B cell sorting and deep B cell receptor sequencing. Notably, we used fluorescently-labeled envelope proteins from each virus to isolate virus-specific B cells from infected peripheral blood. We identified multiple unique B cells positive for both HIV and HCV antigens, which we then expressed as recombinant antibodies to characterize. Intriguingly, we see antibody-mediated neutralization of diverse viral strains, suggesting a common mode of antiviral protection. Matching single cell sequencing and functional to next-generation sequencing data allows for powerful delineation and characterization of virus-specific antibody responses. Furthermore, this study presents the first evidence of antibody cross-reactivity between genetically unrelated viruses, which could have great impact on future vaccine design efforts.
Reprogramming the antigen specificity of B cells using genome-editing technologies
Authors: Gonzalez-Martin Alicia , Andrabi Raiees , Fuller Roberta P. , Murrell Ben , McCoy Laura E. , Porter Katelyn , Huang Deli , Li Wenjuan , Sok Devin , Le Khoa , Briney Bryan , Chateau Morgan , Rogers Geoffrey , Hangartner Lars , Feeney Ann J. , Nemazee David , Cannon Paula , Burton Dennis , Voss James E.
Published: January 2023

HIV infection is a global health concern affecting roughly 37 million people and still no effective vaccine is available for prevention. While rare, HIV broadly neutralizing antibodies (bnAbs) have been isolated from patients. These antibodies have been shown to be protective in animal models if present at the time of challenge in passive transfer experiments. Thus, elicitation of HIV bnAbs is a central goal for vaccine design. These antibodies, however, contain rare features not present in the naïve human antibody repertoire. We have developed a universal genome editing strategy to introduce novel paratopes into the human repertoire. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines and human primary B cells with that from an HIV broadly neutralizing antibody, PG9. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific BCRs. The engineered B cells are subject to class switch recombination and somatic hypermutation to generate HIV-specific BCRs with improved neutralizing breath and potency.
Evolution of the AID/APOBEC family of cytidine deaminases in lampreys
Authors: Hirano Masayuki , Sutoh Yoichi , Das Sabyasachi , Cooper Max
Published: January 2023

Jawless vertebrates, lampreys and hagfish, have antigen receptors termed variable lymphocyte receptors (VLRs), which are composed of leucine-rich repeat (LRR) segments. Three types of VLR genes, VLRA, VLRB and VLRC, are somatically assembled in lymphocytes by a gene conversion-like process. In this process, flanking LRR gene cassettes are randomly and sequentially incorporated in a piece-wise fashion into an incomplete germline VLR gene to generate mature VLR genes.

Activation-induced cytidine deaminase (AID), a member of AID/APOBEC family of cytidine deaminases (CDAs), is a well-known contributor to antigen receptor diversification in jawed vertebrates. We have only limited information about the molecular basis for the assembly and diversification of VLR genes in jawless vertebrates. Two AID/APOBEC-type CDAs, CDA1 and CDA2, have been reported in lampreys. We have previously shown that CDA1 is expressed in VLRA+ and VLRC+ lymphocytes, whereas the CDA2 is expressed in VLRB+ lymphocytes. VLRA and VLRC gene assembly coincide with expression of CDA1 in the gill tip region displaying thymus-equivalent features. Although CDA1 has been shown to induce C to U mutations in bacterial and yeast DNA, mutational activity of CDA2 has not been reported. To find molecules for VLRB gene assembly, we have searched the lamprey genomic database and our transcriptome data from lamprey lymphocytes, and have identified four splice variants of CDA2. Gene expression analysis indicated that all CDA2 splice variants are expressed only in VLRB+ cells. We also found that some of the CDA2 splice variants have AID-like mutational activity in the in vitro system. These results suggest the function and regulation of CDA2 splice variants in VLRB+ cells.
Somatic hypermutation of T cell receptor gamma, delta, and beta chains in nurse sharks
Authors: Ott Jeannine A , Harrison Jenna , Ota Yuko , Flajnik Martin F , Criscitiello Michael F
Published: January 2023

Many γδ T cells typically bind antigen in a manner similar to that of immunoglobulins, recognizing and directly binding small molecules and intact proteins without presentation by classical MHC:Ag complexes. In this way, γδ T cells combine an innate-like immune response with an adaptive recognition strategy, providing both an immediate response to pathogen invasion and an ongoing, adaptive response to inflammation. However, γδ T cells often recombine tissue-specific, restricted sets of genes that have limited junctional diversity. Thus, somatic hypermutation (SHM) presents a useful solution for diversifying these antibody-like T cell receptors (TCR) by fine-tuning ligand recognition or permitting changes within the loci that allow receptors to evolve more rapidly to changing ligand environments. In fact, recent studies in sandbar shark have confirmed definitively that the γ chain locus of γδ T cells employs SHM as a mechanism to generate more diverse γδ TCR, and follow-up studies in dromedary camel indicated that loci of both γ and δ chains use SHM to diversify γδ TCR. In nurse sharks, we found evidence that SHM acts in the thymus, altering the α chain locus of αβ T cells to broaden the primary αβ T cell repertoire. Further, early results suggest that SHM modifies both γ and δ chain loci (but not β chain locus of αβ T cells) and this alteration may occur in the thymus, indicating that this receptor fine-tuning may precede ligand recognition.
CD8+ T Cells require IL-27R for optimal generation of an effector response during influenza infection
Authors: Dunbar Paul , Li Zheng-Rong Tiger , Lobby Jenna , Hayward Sarah L , Lawrence Laurel , Kohlmeier Jacob E
Published: January 2023

Previous reports have demonstrated the many effects of IL-27 on CD4 T cell activation and differentiation, in addition to a role for IL-27 in cancer therapies mediated by cytolytic CD8 T cells. However, as IL-27 is produced by many cell types during infection, and the IL-27 receptor (IL-27R) is widely expressed on NK cells, as well as on activated CD4 and CD8 T cells, the direct effects of IL-27 signaling on CD8 T cell function are less well understood. We used a murine model of influenza infection in combination with co-transfer of congenic wild type (WT) and IL-27R−/− OT-I CD8 T cells to investigate whether IL-27 signaling had a direct effect on virus-specific CD8 T cell function in vivo. We observed a dramatic reduction in the frequency and number of IL-27R−/− OT-I compared to their WT OT-I counterparts within the same host following influenza infection, indicating a CD8 T cell-intrinsic role for IL-27 in the generation of an acute anti-viral response. This defect was observed in the spleen, lung interstitium, airways and the lung-draining mediastinal lymph node, indicating a systemic defect in development of the CD8 T cell effector response in the absence of IL-27R signaling. Both memory precursor and short lived effector cells were reduced in the absence of IL-27R signaling indicating potential defects in both effector and memory generation. We are currently exploring the mechanism of defective effector CD8 T cell generation in the absence of cell-intrinsic IL-27R signaling. Overall, these findings show a cell-intrinsic role for IL-27 signaling in the generation of a robust effector CD8 T cell response following influenza infection.
The Nonlinear Relations that Predict Influenza Viral Dynamics, Host Response, Pathology, and Disease Severity
Authors: Smith Amber M
Published: January 2023

Influenza A viruses cause a significant amount of morbidity and mortality. Understanding how the infection is controlled by host immune responses and how different factors influence severity are critical to combat the infection. During infection, virus increases exponentially, peaks, then declines until resolution. The viral decline is often biphasic, which we previously determined is a consequence of density-dependent infected cell clearance. The second, rapid clearance phase corresponds with the infiltration of CD8 T cells, but how the rate changes with infected cell density and CD8 density is unclear. Further, neither of these kinetics directly correlate to disease severity. Thus, we investigated these relations by infecting mice with influenza A/PR8, simultaneously measuring virus and CD8s, and developing/calibrating a kinetic model. The model predicts that virus resolution is sensitive to T cell expansion, that there is a critical CD8 magnitude below which the infection is significantly prolonged, and that the efficiency of CD8-mediated clearance is dependent on infected cell density. To further examine this finding and validate the model, we quantified infected cells kinetics using histomorphometry. These data showed that the area of lung infected reflects the predicted infected cell dynamics, and that the infection resolution dynamics parallel the relative CD8 magnitude. Our analysis further revealed a nonlinear relation between disease severity and the percent damaged lung. Establishing these critical connections that map the kinetics of virus, infected cells, T cells, lung pathology, and disease severity aids our ability to predict the course of infection, disease progression, and potential complications.
Influenza virus M1 interacts with SLD5 to block host cell cycle and inhibit lung epithelial regeneration
Authors: Fang Min , Zhu Li , Zhao Wenming
Published: January 2023

Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here we used a yeast two-hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 inhibited influenza virus replication. Transient, stable or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Importantly, SLD5 partly rescued M1 expression- or influenza virus infection-induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared to wild type mice. Therefore, influenza virus M1 inhibits host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus-host interaction.
BCL6 modulates tissue neutrophil survival and exacerbates pulmonary inflammation following influenza infection
Authors: Sun Jie , Zhu Bibo
Published: January 2023

B-cell lymphoma 6 (BCL6) is a transcriptional repressor that is best known for its pivotal function in governing follicular T helper cell and germinal center B cell responses. However, its role in innate anti-viral responses is only now being appreciated. Here we find that BCL6 deficiency in myeloid cells exhibited drastically enhanced host resistance to severe influenza infection, which was independent of IFNAR1 signaling. In contrary to the notion that BCL6 functions to suppress innate inflammation, we find that myeloid BCL6 deficiency diminished lung inflammation without affecting viral loads. Using a series of Cre-transgenic, reporter and knockout mouse lines, we demonstrate that BCL6 deficiency in neutrophils, but not in monocytes or macrophages, attenuated host inflammation and morbidity following influenza infection. Mechanistically, BCL6 disruption resulted in increased expression of a number of genes involved in cellular apoptosis in neutrophils. Consequently, BCL6 deficiency promoted neutrophil apoptosis, specifically at the site of infection. Furthermore, partial neutrophil depletion leaded to diminished pulmonary inflammation and decreased host morbidity. Our results have revealed a previously unappreciated role of BCL6 in modulating neutrophil function and apoptosis at the infected lungs for the regulation of host disease development following influenza infection. Furthermore, our studies indicate that neutrophil survival is tightly regulated in a tissue-specific fashion, thereby influencing inflammation and immunopathology development during acute respiratory viral infection.