CD4 Helper Research Articles

Abstract 1660: Humanization of patient derived xenograft (PDX) cancer model mice with peripheral blood mononuclear cells (PBMCs)

Abstract Immunocompetent transgenic mouse models have for decades served as valuable tools to address the effect of oncogenes and tumor suppressors. Immunodeficient mice have been used to establish patient derived xenograft (PDX) models harboring human tumors. These types of models have been used to study cancer initiation and progression as well as preclinical evaluation of anticancer drugs. One major limiting factor for both these model systems is the immune system; the PDX models lack an immune system and the immune system in immunocompetent mouse models display differences compared to the human immune system. Thus, in cancer research there is a need for preclinical models to study the influence of human immune cells on tumor progression and response to cancer therapies. We have established and optimized protocols for intravenous (IV) or intraperitoneal (IP) injections of isolated human PBMCs, to generate humanized mice harboring human immune cells. Our protocols do not require irradiation or busulfan pretreatment of the animals. Flow cytometry analyses showed that mainly T helper cells, CD4+, and cytotoxic T cells, CD8+, were present in the humanized mice. Immunohistochemistry (IHC) analyses of humanized PDX cancer model mice showed that human lymphocytes were present in the tumor periphery and some very few cells within the tumor, in addition to the human leukocytes in the liver and spleen. The growth rates of the PDX tumors were not affected by the humanization. Our protocols require minimal preparations of the animals and generate humanized PDX mice harboring human lymphocytes locating in close proximity of, as well as inside the PDX tumors. The model systems are suitable for preclinical studies of human, adaptive immune responses in tumor progression and cancer therapies. Citation Format: Jens Henrik Norum, Dag Josefsen, Gunnar Kvalheim, Olav Engebråten, Therese Sørlie, Gunhild M. Mælandsmo. Humanization of patient derived xenograft (PDX) cancer model mice with peripheral blood mononuclear cells (PBMCs) [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 1660. doi:10.1158/1538-7445.AM2017-1660

Abstract 3817: Deciphering the immune system in small intestine: Multiplex strategies for inflammatory bowel disease

Abstract Introduction Inflammatory bowel disease (IBD) can affect the lower part of the small intestine (ileum) and the colon. It can, however, occur in any part of the large intestine, small intestine, or stomach. Recent evidence suggests that several factors may tip the balance between homeostasis and intestinal inflammation, presenting future challenges for the development of new therapies for IBD. In this study, we use normal small intestine as a model to identify multiple immune targets using mouse and rabbit monoclonal antibodies. Peyer Patches are lymphoid tissues in the wall of the small intestine that are involved in the development of immunity to antigens presented in that milieu. Small intestine and cases of IBD were evaluated as a potential model using immunohistochemistry (IHC) to identify categories of immune cells including cytotoxic T-cells, costimulatory cells (agonists), T-regulatory cells (TREGs), T-cell effector cells (regulators) and checkpoint inhibitors. cases of IBD were also examined for the utilization of single and double/triple stains. Design Formalin-fixed paraffin-embedded cases of small intestine and IBD were cut to 4 micron thickness and stained with H&E. Mouse and rabbit monoclonal antibodies including CD56 (natural killer cells), CD163 (macrophages), CD8 and CD4 (cytotoxic T-cells and helper T- cells), CD103, OX40 and CD137 (costimulatory), FOXP3 and LAG3 (TREGs), T-bet and RORγT (T-cell effectors), and PD-1 and PD-L1 (immune checkpoints) were tittered for optimization and evaluated for IHC. Several IHC multiplex stains (double and triple stains) were also developed and compared with the relevant single stains. Results Single, double and triple stains utilizing CD56, CD163, CD4, CD8, CD103, OX40, CD137, FOXP3, LAG3, T-Bet, RORγT, PD-1 and PD-L1 antibodies were successfully established using small intestine as staining model for IBD cases. All single stains versus double and triple stains gave equivalent results. Double stains and/or co-expression of CD8 + CD103, CD8 + PD-1, and triple stain CD8 + PD-1 + FOXP3 were easily visualized with blue, brown, red or black chromogen staining. Conclusion Small intestine is a good model to develop single or multiplex stains for a host of immune cell targets. Enhanced visualization of these targets is possible with the use of double and triple stains; thus, this technique may help facilitate acquisition of important prognostic information and help support new strategies for inflammatory bowel disease in targeted therapeutic treatment. Note: This abstract was not presented at the meeting. Citation Format: David Altree-Tacha, Wei Wei Yuan, George Yang. Deciphering the immune system in small intestine: Multiplex strategies for inflammatory bowel disease [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 3817. doi:10.1158/1538-7445.AM2017-3817

Allergen-specific immunotherapy modulates the balance of circulating Tfh and Tfr cells

Abstract Follicular helper T cells (Tfh cells) are a CD4 T cell subset essential for germinal center formation and B cell responses, although their role in allergy and allergen-specific immunotherapy (AIT) is unclear. Here, we show that AIT-treated patients have a higher ratio of regulatory Tfh cells (Tfr) to follicular T cells (Tfh) and hypothesize an IL-2 dependent mechanism.

CXCR5-Dependent Entry of CD8 T Cells into Rhesus Macaque B-Cell Follicles Achieved through T-Cell Engineering.

Follicular helper CD4 T cells, TFH, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXC chemokine receptor 5 (CXCR5), the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing human CXCR5 (CD8hCXCR5) exhibited ligand-specific signaling and chemotaxis in vitro Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8hCXCR5 and untransduced CD8 T cells and necropsied 48 h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8hCXCR5 T cells were present throughout the follicles with some observed near infected TFH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication.IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, TFH, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles, and those that do fail to robustly control viral replication in the TFH population. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on TFH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

MAIT cells (CD8+CD161++TCR7.2++) are functionally impaired during chronic SHIV infection.

Abstract Mucosa-associated invariant T-cells (MAIT) are abundant in humans and recognize bacterial ligands. Here, we have studied the distribution and function of MAIT cells during chronic SHIV infection using rhesus macaques (RM). Two groups of RM, healthy and SHIV infected were studied. lymphocytes from various tissues were analysed for MAIT (TCR7.2++ CD161++) cells using multicolor flow cytometry and characterized for their distribution, phenotype and function during chronic SHIV infection. Similar to human, we found significant fraction of (~2%) RM CD8 T cells co-express MAIT cell markers TCR7.2, CD161, IL-18R, CCR6 and display central memory (CCR7+CD45RA−) phenotype. The frequency of MAIT cells were decreased during chronic SHIV infection and tissue analysis showed a significant enrichment of MAIT cells in liver (~8%) and BAL (3%) than in blood (1%), spleen(0.5%), lymph node(0.13%) and gut (0.2%). Importantly, during chronic SHIV infection more than 80% of MAIT cells expressed T cell exhaustion marker PD-1 in blood and the level of expression was higher (>95%) in tissue resident CD69+MAIT cells. These cells lack proliferating capacity (Ki-67) and cytokine production (IFN- g and IL-17) in the blood, suggesting their functional impairment. The preserved blood and gut MAIT cells correlate inversely with plasma viral RNA levels and associate directly with gut CD4 T cell levels, suggesting that CD4 help maintains MAIT cell frequencies in the gut. These data demonstrate that MAIT cells are decreased in blood and are functionally exhausted during chronic SHIV infection. Future studies focused on blocking the PD-1 pathway to retrive MAIT cell exhaustion during chronic SHIV/HIV infection may yield therapeutic benefit to HIV/SHIV infection.

Antigen-presenting tumor B cells impact the phenotype of CD4 tumor infiltrating T cells in lung cancer patients

Abstract The focus of immunotherapy has been on CD8 and CD4 tumor infiltrating lymphocytes (TILs), however, tumor infiltrating B cells (TIL-Bs) are understudied with no focus on their role as antigen presenting cells. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs. Using un-manipulated, primary human B cells from fresh tumor, we generated a specific in vitro antigen presentation assay and observed three types of CD4 TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4 TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required autologous tumor lysate to elicit a CD4 TIL response, and there were patient CD4 TILs that did not respond to antigen presentation. Exhausted B cells have been described in chronic infection i.e. HIV, and thus, to parallel our studies to a model of chronic infection, we analyzed activated and exhausted TIL-Bs with our antigen presentation assay. If TIL-Bs were activated (HLADR+CD69+CD27+CD21+), the resultant CD4 TILs were T helper (anti-tumor) CD4 T cells and if the TIL-Bs were exhausted (HLADR+CD69+CD27−CD21−), the resultant CD4 TILs were T regulatory cells (pro-tumor). These data suggest that TIL-Bs influence CD4 TILs in NSCLC patient tumors. In conclusion, determining if TIL-Bs are activated or exhausted in NSCLC patients will determine the extent of their anti-tumor function in human cancer. Ultimately, results from this study will dictate how to target TIL-Bs in future immunotherapies.

A subset of NK1.1+ follicular helper CD4+ T cells promotes early extrafollicular plasmablast production during Plasmodium yoelii infection

Abstract Early plasmablast induction is a hallmark of Plasmodium infection, including P. yoelii, and is thought to be essential for control of acute parasite burden. Although long understood to be a T-cell dependent phenomenon, control of early plasmablast differentiation in response to Plasmodium is poorly understood. Here, we identify a subset of CD4+ T cells that express the innate NK cell marker NK1.1 as being critical for early plasmablast and parasite-specific Ab production. Interestingly, NK1.1+ CD4+ T cells arise from conventional, naive NK1.1− CD4+ T cells and their induction is independent of CD1d but critically reliant on MHC-II. Early NK1.1+ CD4+ T cells express more ICOS than conventional (NK1.1−) CD4+ T cells and subsequently expand more rapidly and give rise to PD-1high follicular helper T (Tfh) cells more frequently than conventional CD4+ T cells. Further study of this population revealed that NK1.1+ Tfh cells are more likely to be complexed with plasmablasts than conventional Tfh cells. Moreover, NK1.1+ Tfh cells interact almost exclusively with PD-L1high plasmablasts and more readily express markers indicative of apoptosis than conventional Tfh cells. Ultimately, depletion of NK1.1+ cells significantly impairs class-switched parasite-specific antibody production during early P. yoelii infection. Together, these data suggest NK1.1+ Tfh cells are essential for driving early plasmablast induction during Plasmodium infection and may represent a unique Tfh cell subset whose modulation could promote effective vaccine design.

HetIL-15 treatment of rhesus macaques is safe and systemically increases effector cytotoxic lymphocytes that infiltrate the lymph nodes.

Abstract Background Heterodimeric interleukin-15 (hetIL-15) is presently evaluated in humans for the treatment of cancer. hetIL-15 activates and expands cytotoxic T and NK cells, which suggests that the cytokine could be useful for treating diseases such as malignancies and HIV infection. Methods Rhesus macaques received six s.c. injections of hetIL-15 over 2 weeks using increasing doses of cytokine (step-dosing). Animals were sacrificed after treatment and hetIL-15 effects on lymphocytes isolated from necropsy tissues were monitored by multiparametric flow cytometry and quantitative multiplexed confocal microscopy imaging (Histo-cytometry). Results This regimen was found to be safe in macaques and resulted in systemic proliferation and expansion of CD8+ and NK cells with higher granzyme B content. These expanded cells were found in both effector sites, such as liver, vagina and rectum, and secondary lymphoid tissues. A significant increase in effector memory CD8+ T cells was found in peripheral lymph nodes (LN) from all hetIL-15-treated macaques. Imaging analysis by Histo-cytometry revealed that effector CD8+ T cells infiltrated the B cell follicles where chronically infected follicular helper CD4+ T cells are located. Conclusions Step-dose administration of hetIL-15 is a well-tolerated regimen that results in systemic activation and expansion of cytotoxic leukocytes that infiltrate areas where chronic HIV-infected cells reside. These results suggest that hetIL-15 could be useful in disrupting or eliminating long-term viral reservoirs in HIV-1 infected individuals, contributing to a functional cure of the infection. Work assessing the impact of hetIL-15 on the size of the viral reservoir in sampled LN is currently ongoing.

Augmenting Ag presentation at the “CD4 memory checkpoint” can overcome age-associated “defects” in CD4 helper response and enhance vaccination.

Abstract Vaccines are the most efficient approach to prevent infection and transmission of viruses such as influenza, but they are inefficient in the elderly. Age-associated functional defects in naive CD4 T cells, lead to few Tfh and poor help for B cells and thus to reduced long-lived Ab and memory CD4 T cells. Therefore, a vaccination strategy that would enhance the response of aged CD4 T cells could promote B and T cell memory generation and more effective protection. We found CD4 effectors generated by influenza A virus (IAV) made very few memory cells if they could not make autocrine IL-2 and that such IL-2 production depended on cognate Ag recognition at the effector stage to drive transition to memory, defining what we call the ‘memory checkpoint’. Full differentiation of CD4 effectors to Tfh/GC-Tfh effectors also requires such Ag recognition at this checkpoint. Here we ask if providing adult or aged CD4 T cells with Ag/APC at the ‘memory checkpoint’ can augment the CD4 and Ab response to inactivated IAV (IIV) vaccine. We use a reductionist in vivo model with an irrelevant TcR transgenic host, where we can add the IAV-specific naïve CD4 T cells (young or aged from TcR transgenic donor) and vaccinate with inactivated IAV as the vaccine. We introduce Ag at the memory checkpoint. When with either young adult or aged CD4+ T cells, again recognize cognate Ag at the ‘memory checkpoint’, they yield more IAV-specific IgG memory B cells (MBCs), IgG-secreting long-lived plasma cells (LLPCs) and more tissue resident memory CD4 T cells. This suggests novel strategies that augment Ag presentation at this CD4 checkpoint may improve vaccine efficacy in both young and elderly.

Dissecting T follicular helper cell development in vivo using CRISPR

Abstract T follicular helper (Tfh) cells are specialized CD4 helper T cells that signal B cells to produce high affinity antibodies and provide long-term humoral immunity. Identifying genes that govern Tfh differentiation and function could provide targets for improving vaccines and therapies for autoimmunity. However, Tfh differentiation is only partially understood, and not fully recapitulated in vitro. To discover novel Tfh-regulating genes, we developed a CRISPR-based functional genetic system to mutate genes in primary mouse T cells, and measured the effects on Tfh cells in vivo. Transgenic Cas9-expressing mouse CD4 T cells were transduced in vitro with an optimized retroviral vector encoding an sgRNA and GFP. With a guide targeting Tcf7 (encoding a Tfh-essential transcription factor Tcf1), >90% cells could be transduced; up to >80% of GFP+ cells were Tcf1neg by flow and mutational analyses. To evaluate the functional consequence of CRISPR editing, we adoptively transferred virus-specific CD4 T cells that were transduced with either a control or Tcf7 sgRNA construct into wild-type hosts. After viral challenge, control T cells differentiated into both Tfh and non-Tfh cells, while very few Tcf7-edited T cells became Tfh cells. Similarly, guides against the Tfh master transcription factor Bcl6 abrogated Tfh differentiation in vivo. Next, we constructed sgRNA libraries of up to 400 guides, targeting up to 80 genes, including ones implicated in primary immunodeficiencies and druggable targets. NGS analysis of T cells post-viral challenge revealed depletion of multiple guides targeting known Tfh-required genes in the Tfh vs non-Tfh populations, validating this approach. We are currently following up on potential hits from these screens.

The Effect of Antiretroviral Naïve HIV-1 Infection on the Ability of Natural Killer Cells to Produce IFN-γ upon Exposure to Plasmodium falciparum-Infected Erythrocytes

Background: In sub-Saharan Africa, intense perennial Plasmodium species transmission coincides with areas of high prevalence of the human immunodeficiency virus type 1 (HIV) infection. This implies that antiretroviral naïve HIV-infected people living within these regions are repeatedly exposed to Plasmodium species infection and consequently malaria. Natural killer (NK) cells are known to contribute to malaria immunity through the production of IFN-γ after exposure to Plasmodium falciparum-infected erythrocytes (infected red blood cells [iRBC]). However, in antiretroviral naïve HIV-1 infection, these functions could be impaired. In this study we assess the ability of NK cells from antiretroviral naïve HIV-1-infected people to respond to iRBC. Method: Magnetically sorted NK cells from antiretroviral naïve HIV-1-infected people were tested for their ability to respond to iRBC following in vitro coculture. NK cell IFN-γ production after coculture was measured through multiparametric flow cytometry analysis. Results: Our data show a significant reduction (p = 0.03) in IFN-γ production by NK cells from antiretroviral naïve HIV-1-infected people after coculture with iRBCs. This was in contrast to the NK cell response from healthy controls, which demonstrated elevated IFN-γ production. NK cell IFN-γ production from untreated HIV-1-infected participants correlated inversely with the viral load (r = -0.5, p = 0.02) and positively with total helper CD4⁺ T-cell count (r = 0.4, p = 0.04). Thus, antiretroviral naïve HIV-1 infection can dampen NK cell-mediated immunity to P. falciparum infection in malaria-intense regions. This could in effect escalate morbidity and mortality in people chronically infected with HIV-1.

IL-6 Production by TLR-Activated APC Broadly Enhances Aged Cognate CD4 Helper and B Cell Antibody Responses In Vivo.

Naive CD4 T cell responses, especially their ability to help B cell responses, become compromised with aging. We find that using APC pretreated ex vivo with TLR agonists, polyinosinic-polycytidylic acid and CpG, to prime naive CD4 T cells in vivo, restores their ability to expand and become germinal center T follicular helpers and enhances B cell IgG Ab production. Enhanced helper responses are dependent on IL-6 production by the activated APC. Aged naive CD4 T cells respond suboptimally to IL-6 compared with young cells, such that higher doses are required to induce comparable signaling. Preactivating APC overcomes this deficiency. Responses of young CD4 T cells are also enhanced by preactivating APC with similar effects but with only partial IL-6 dependency. Strikingly, introducing just the activated APC into aged mice significantly enhances otherwise compromised Ab production to inactivated influenza vaccine. These findings reveal a central role for the production of IL-6 by APC during initial cognate interactions in the generation of effective CD4 T cell help, which becomes greater with age. Without APC activation, aging CD4 T cell responses shift toward IL-6-independent Th1 and CD4 cytotoxic Th cell responses. Thus, strategies that specifically activate and provide Ag to APC could potentially enhance Ab-mediated protection in vaccine responses.

Immune Class Regulation and Its Medical Significance Part II of a Report of a Workshop on Foundational Concepts of Immune Regulation.

We discussed different proposals for how the nature of the Th1/Th2 phenotype of an immune response is determined, and favoured one, the Threshold Hypothesis, as plausible and so useful as the basis for further discussions. The activation of a target CD4 T cell can be facilitated by helper CD4 T cells when the CD4 T cells interact via an antigen-presenting cell. The Threshold Hypothesis states that tentative and robust antigen-mediated CD4 T cell cooperation results in the target CD4 T cell, respectively giving rise, upon activation, to Th1 and Th2 cells. We primarily discussed four topics. We briefly discussed in the background section certain limitations of the Th1/Th2 paradigm in understanding immune class regulation, and the remarkable anti-inflammatory properties of human IgG4 antibody. Secondly, we assessed the role of class II MHC molecules in determining the number of mature CD4 T cells and so affecting the Th1/Th2 phenotype of immune responses. We also discussed the controversial role of CD8 T cells in affecting the Th1/Th2 phenotype of responses to MHC and other antigens, and the potential role of their relative scarcity in neonates in biasing responses towards an antibody, Th2 mode. Lastly, we examined the regulation of the Th1/Th2 phenotype of both primary and ongoing immune responses in the context of the intriguing proposal that antigen initially generates different classes/subclasses of immunity and then selects, by a feedback mechanism, the most effective class. We found this interesting idea difficult to reconcile with various observations.

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

Immune control of viral infections is heavily dependent on helper CD4+ T cell function. However, the understanding of the contribution of HIV-specific CD4+ T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4+ T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4+ T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4+ T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4+ T cells in HIV controllers than progressors (P = 0.0001), and these expanded Gag-specific CD4+ T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control (r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4+ T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4+ T cell responses in natural infections.IMPORTANCE Increasing evidence suggests that virus-specific CD4+ T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4+ T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV infections worldwide. Understanding the contribution of HIV-specific CD4+ T cell responses in clade C infection is particularly important for developing vaccines that would be efficacious in sub-Saharan Africa, where clade C infection is dominant. Here, we employed MHC class II tetramers designed to immunodominant Gag epitopes and used them to characterize CD4+ T cell responses in HIV-1 clade C infection. Our results demonstrate an association between the frequency of HIV-specific CD4+ T cell responses targeting an immunodominant DRB1*11-Gag41 complex and HIV control, highlighting the important contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infections.

Evaluating the antitumor role of B cells in patients with non-small cell lung cancer.

75 Background: The focus of immunotherapy has been on CD8 and CD4 tumor infiltrating lymphocytes (TILs), however, tumor infiltrating B cells (TIL-Bs) have been reported in tertiary lymphoid structures (TLS) with CD4 TILs, and both TIL-Bs and TLS correlate with NSCLC patient survival. While TIL-Bs have been identified in NSCLC patients, their function in the tumor microenvironment has been understudied with no focus on their role as antigen presenting cells (APCs) and their influence on CD8 and CD4 TILs. We hypothesize that TIL-Bs help generate potent, long-term immune responses against cancer by presenting tumor antigens to CD4 TILs. Methods: We used freshly isolated B cells from NSCLC patient tumor and tumor adjacent tissue to determine TIL-B function. We generated a specific in vitro antigen presentation assay to determine APC function and immunoregulation by TIL-Bs, and we correlated our findings with tumor-free survival. Results: We observed that the total number of B cells at the site of the tumor versus the tumor-adjacent tissue was increased compared to other immune subsets. Further, we observed three types of CD4 TIL responses when TIL-Bs presented autologous tumor antigens. There were activated responder CD4 TILs that proliferated when combined with TIL-Bs alone, which indicates stimulation with endogenous tumor antigens. There were antigen-associated responders that required exogenous autologous tumor lysate to elicit a CD4 TIL response, and there were patient CD4 TILs that did not respond to antigen presentation by TIL-Bs. Within the activated and antigen-associated responders, the TIL-B phenotype influenced the CD4 TIL phenotype; if the TIL-Bs were activated (CD69+CD27+CD21+), the CD4 TILs were T helper (anti-tumor) CD4 T cells and if the TIL-Bs were exhausted (CD69-CD27-CD21-), the CD4 TILs were T regulatory cells (pro-tumor). These data suggest that TIL-Bs influence the phenotype and function of CD4 TILs in NSCLC patient tumors. Conclusions: In conclusion, determining if TIL-Bs are activated or exhausted in NSCLC patients will determine the extent of their anti-tumor function in cancer. Ultimately, results from this study will help predict how to target TIL-B functions in future immunotherapies for NSCLC patients.

Human CD4+ T Cell Responses to an Attenuated Tetravalent Dengue Vaccine Parallel Those Induced by Natural Infection in Magnitude, HLA Restriction, and Antigen Specificity.

Dengue virus (DENV) is responsible for growing numbers of infections worldwide and has proven to be a significant challenge for vaccine development. We previously demonstrated that CD8+ T cell responses elicited by a dengue live attenuated virus (DLAV) vaccine resemble those observed after natural infection. In this study, we screened peripheral blood mononuclear cells (PBMCs) from donors vaccinated with a tetravalent DLAV vaccine (TV005) with pools of dengue virus-derived predicted major histocompatibility complex (MHC) class II binding peptides. The definition of CD4+ T cell responses after live vaccination is important because CD4+ T cells are known contributors to host immunity, including cytokine production, help for CD8+ T and B cells, and direct cytotoxicity against infected cells. While responses to all antigens were observed, DENV-specific CD4+ T cells were focused predominantly on the capsid and nonstructural NS3 and NS5 antigens. Importantly, CD4+ T cell responses in vaccinees were similar in magnitude and breadth to those after natural infection, recognized the same antigen hierarchy, and had similar profiles of HLA restriction. We conclude that TV005 vaccination has the capacity to elicit CD4+ cell responses closely mirroring those observed in a population associated with natural immunity.IMPORTANCE The development of effective vaccination strategies against dengue virus infection is of high global public health interest. Here we study the CD4 T cell responses elicited by a tetravalent live attenuated dengue vaccine and show that they resemble responses seen in humans naturally exposed to dengue virus. This is an important issue, since it is likely that optimal immunity induced by a vaccine requires induction of CD4+ responses against the same antigens as those recognized as dominant in natural infection. Detailed knowledge of the T cell response may further contribute to the identification of robust correlates of protection against dengue virus.

Quantification of Residual Germinal Center Activity and HIV-1 DNA and RNA Levels Using Fine Needle Biopsies of Lymph Nodes During Antiretroviral Therapy.

HIV-1 reservoirs are most often studied in peripheral blood (PB), but not all lymphocytes recirculate, particularly T follicular helper (Tfh) CD4+ T cells, as well as germinal center (GC) B cells, in lymph nodes (LNs). Ultrasound-guided fine needle biopsies (FNBs) from inguinal LNs and PB samples were obtained from 10 healthy controls (HCs) and 21 HIV-1-infected subjects [11 antiretroviral therapy (ART) naive and 10 on ART]. Tfh cells and GC B cells were enumerated by flow cytometry. HIV-1 DNA and cell-associated (CA) RNA levels in LNs and PB were quantified by real-time polymerase chain reaction. FNBs were obtained without adverse events. Tfh cells and GC B cells were highly elevated in ART-naive subjects, with a median GC B cell count >300-fold higher than HCs, but also remained higher in 4 out of the 10 subjects on ART. GC B cell counts and Tfh cell counts were highly correlated with each other, and also with activated T cells in LNs but not in blood. Levels of HIV-1 DNA and CA RNA viral burden in highly purified CD4+ T cells from FNBs were significantly elevated compared with those in CD4+ T cells from PB in the ART-naive group, but only trended toward an increase in the ART patients. FNBs enabled minimally invasive access to, and parallel measurement of residual activated T and B cells and viral burden within LNs in HIV-1-infected patients. These FNBs revealed significant GC activity that was not apparent from corresponding PB samples.

MTOR Promotes Antiviral Humoral Immunity by Differentially Regulating CD4 Helper T Cell and B Cell Responses.

mTOR is a serine/threonine kinase involved in a variety of cellular activities. Although its specific inhibitor, rapamycin, is currently used as an immunosuppressive drug in transplant patients, it has been reported that rapamycin can also stimulate pathogen-specific cellular immunity in certain circumstances. However, whether and how mTOR regulates humoral immunity are not well understood. Here we found that rapamycin treatment predominantly inhibited GC B cell responses during viral infection and that this led to biased helper CD4 T cell differentiation as well as impaired antibody responses. These findings suggest that inhibition of B cell responses by rapamycin may play an important role in regulation of allograft-specific antibody responses to prevent organ rejection in transplant recipients. Our results also show that consideration of antibody responses is required in cases where rapamycin is used to stimulate vaccine-induced immunity.

Nanoparticles decorated with viral antigens are more immunogenic at low surface density

There is an urgent need to develop protective vaccines for high priority viral pathogens. One approach known to enhance immune responses to viral proteins is to display them on a nanoparticle (NP) scaffold. However, little is known about the effect of protein density on the B cell response to antigens displayed on NPs. To address this question HIV-1 Envelope (Env) and influenza hemagglutinin (HA) were displayed on a polystyrene-based NP scaffold at various densities - corresponding to mean antigen distances that span the range encountered on naturally occurring virions. Our studies revealed that NPs displaying lower densities of Env or HA more efficiently stimulated antigen-specific B cells in vitro, as measured by calcium flux, than did NPs displaying higher antigen densities. Similarly, NPs displaying a low density of Env or HA also elicited higher titers of antigen-specific serum IgG in immunized BALB/c mice (including elevated titers of hemagglutination-inhibiting antibodies), as well as an increased frequency of antigen-specific antibody secreting cells in the lymph node, spleen and bone marrow. Importantly, our studies showed that the enhanced B cell response elicited by the lower density NPs is likely secondary to more efficient development of follicular helper CD4 T cells and germinal center B cells. These findings demonstrate that the density of antigen on a NP scaffold is a critical determinant of the humoral immune response elicited, and that high density display does not always result in an optimal response.

Optimization of Peptide Vaccines to Induce Robust Antitumor CD4 T-cell Responses.

Substantial evidence indicates that immunotherapy is a feasible and effective approach for the treatment of numerous types of cancer. Among various immunotherapy options, peptide vaccines to generate antitumor T cells appear as promising candidates, because of their cost effectiveness and ease of implementation. Nevertheless, most peptide vaccines are notorious for being weekly immunogenic and, thus, optimization of the vaccination strategy is essential to achieve therapeutic effectiveness. In addition, effective peptide vaccines must stimulate both CD8 cytotoxic and CD4 helper T lymphocytes. Our group has been successful in designing effective peptide vaccination strategies for inducing CD8 T-cell responses in mouse tumor models. Here, we describe a somewhat similar, but distinct, peptide vaccination strategy capable of generating vast CD4 T-cell responses by combining synthetic peptides with toll-like receptor (TLR) agonists and OX40/CD40 costimulation. This vaccination strategy was efficient in overcoming immune tolerance to a self-tumor-associated antigen and generated significant antitumor effects in a mouse model of malignant melanoma. The optimized peptide vaccine also allowed the expansion of adoptively transferred CD4 T cells without the need for lymphodepletion and IL2 administration, generating effective antimelanoma responses through the enhancement of proliferative and antiapoptotic activities of CD4 T cells. These results have practical implications in the design of more effective T-cell-based immunotherapies. Cancer Immunol Res; 5(1); 72-83. ©2016 AACR.