Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

HIV-1 induction of tolerogenic dendritic cells is mediated by cellular interaction with suppressive T cells.

HIV-1 infection gives rise to a multi-layered immune impairment in most infected individuals. The chronic presence of HIV-1 during the priming and activation of T cells by dendritic cells (DCs) promotes the expansion of suppressive T cells in a contact-dependent manner. The mechanism behind the T cell side of this HIV-induced impairment is well studied, whereas little is known about the reverse effects exerted on the DCs. Herein we assessed the phenotype and transcriptome profile of mature DCs that have been in contact with suppressive T cells. The HIV exposed DCs from cocultures between DCs and T cells resulted in a more tolerogenic phenotype with increased expression of e.g., PDL1, Gal-9, HVEM, and B7H3, mediated by interaction with T cells. Transcriptomic analysis of the DCs separated from the DC-T cell coculture revealed a type I IFN response profile as well as an activation of pathways involved in T cell exhaustion. Taken together, our data indicate that the prolonged and strong type I IFN signaling in DCs, induced by the presence of HIV during DC-T cell cross talk, could play an important role in the induction of tolerogenic DCs and suppressed immune responses seen in HIV-1 infected individuals.

Complement opsonization of HIV affects primary infection of human colorectal mucosa and subsequent activation of T cells.

HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complement-opsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.

HSV-2 Cellular Programming Enables Productive HIV Infection in Dendritic Cells.

Genital herpes is a common sexually transmitted infection caused by herpes simplex virus type 2 (HSV-2). Genital herpes significantly enhances the acquisition and transmission of HIV-1 by creating a microenvironment that supports HIV infection in the host. Dendritic cells (DCs) represent one of the first innate cell types that encounter HIV-1 and HSV-2 in the genital mucosa. HSV-2 infection has been shown to modulate DCs, rendering them more receptive to HIV infection. Here, we investigated the potential mechanisms underlying HSV-2-mediated augmentation of HIV-1 infection. We demonstrated that the presence of HSV-2 enhanced productive HIV-1 infection of DCs and boosted inflammatory and antiviral responses. The HSV-2 augmented HIV-1 infection required intact HSV-2 DNA, but not active HSV-2 DNA replication. Furthermore, the augmented HIV infection of DCs involved the cGAS-STING pathway. Interestingly, we could not see any involvement of TLR2 or TLR3 nor suppression of infection by IFN-ß production. The conditioning by HSV-2 in dual exposed DCs decreased protein expression of IFI16, cGAS, STING, and TBK1, which is associated with signaling through the STING pathway. Dual exposure to HSV-2 and HIV-1 gave decreased levels of several HIV-1 restriction factors, especially SAMHD1, TREX1, and APOBEC3G. Activation of the STING pathway in DCs by exposure to both HSV-2 and HIV-1 most likely led to the proteolytic degradation of the HIV-1 restriction factors SAMHD1, TREX1, and APOBEC3G, which should release their normal restriction of HIV infection in DCs. This released their normal restriction of HIV infection in DCs. We showed that HSV-2 reprogramming of cellular signaling pathways and protein expression levels in the DCs provided a setting where HIV-1 can establish a higher productive infection in the DCs. In conclusion, HSV-2 reprogramming opens up DCs for HIV-1 infection and creates a microenvironment favoring HIV-1 transmission.

Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India.

BACKGROUND: Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. METHODOLOGY/PRINCIPAL FINDINGS: We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. CONCLUSIONS/SIGNIFICANCE: ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response. It can therefore, be deployed widely for interventions in disease management of VL particularly in poor resource outskirts.

Treatment of visceral leishmaniasis: anomalous pricing and distribution of AmBisome and emergence of an indigenous liposomal amphotericin B, FUNGISOME.

Visceral leishmaniasis (VL) is one of the severest forms of parasite borne diseases worldwide with a mortality rate second only to malaria. Treatment of VL patients with currently available chemotherapeutic agents poses problems of large scale failure, toxicity, prolonged hospitalization time, high treatment cost and drug resistance. However, most of these problems can be overcome by the use of liposomal formulations of Amphotericin B (L-AmB). Of the two L-AmBs currently available in Indian market, AmBisome is imported and FUNGISOME is indigenous. Initially AmBisome remained exorbitantly costly and therefore inaccessible to most of the VL patients. However, with the launch of FUNGISOME in India, Gilead in agreement with WHO started a donation program of AmBisome in developing countries through a slashed price of US $18 per vial. The price reduction is, however, restricted to clinical trials thus eluding majority of the VL patients. In fact, India was not included in this program and AmBisome was sold in Indian market at prices higher than the WHO proposed price of US $18 per vial. FUNGISOME, on the other hand, produced consistently good results against VL both clinically and experimentally. In the context of unavailability and price anomaly of AmBisome, successful emergence of FUNGISOME could mark it as the major L-AmB against VL.

A Lipid Based Antigen Delivery System Efficiently Facilitates MHC Class-I Antigen Presentation in Dendritic Cells to Stimulate CD8(+) T Cells.

The most effective strategy for protection against intracellular infections such as Leishmania is vaccination with live parasites. Use of recombinant proteins avoids the risks associated with live vaccines. However, due to low immunogenicity, they fail to trigger T cell responses particularly of CD8(+) cells requisite for persistent immunity. Previously we showed the importance of protein entrapment in cationic liposomes and MPL as adjuvant for elicitation of CD4(+) and CD8(+) T cell responses for long-term protection. In this study we investigated the role of cationic liposomes on maturation and antigen presentation capacity of dendritic cells (DCs). We observed that cationic liposomes were taken up very efficiently by DCs and transported to different cellular sites. DCs activated with liposomal rgp63 led to efficient presentation of antigen to specific CD4(+) and CD8(+) T cells. Furthermore, lymphoid CD8(+) T cells from liposomal rgp63 immunized mice demonstrated better proliferative ability when co-cultured ex vivo with stimulated DCs. Addition of MPL to vaccine enhanced the antigen presentation by DCs and induced more efficient antigen specific CD8(+) T cell responses when compared to free and liposomal antigen. These liposomal formulations presented to CD8(+) T cells through TAP-dependent MHC-I pathway offer new possibilities for a safe subunit vaccine.

Induction of IL-10 and TGFß from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani.

BACKGROUND: Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFß and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFß and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFß to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFß respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4+CD25+ (r = 0.55), CD4+CD25hi (r = 0.61) as well as percentages of CD4+CD25+FoxP3+ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4+CD25+ and CD4+CD25+FoxP3+ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFß in comparison to corresponding CD25- T cells. CONCLUSIONS/SIGNIFICANCE: Our findings shed some light on source(s) of TGFß and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4+CD25+FoxP3+ Treg cells as a source of IL-10 and TGFß during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.

Therapeutic and immunomodulatory activities of short-course treatment of murine visceral leishmaniasis with KALSOME™10, a new liposomal amphotericin B.

BACKGROUND: Visceral leishmaniasis (VL), a potentially fatal disease, is most prevalent in the Indian subcontinent, East Africa and South America. Since the conventional antileishmanial drugs have many limitations we evaluated a new ergosterol rich liposomal amphotericin B formulation, KALSOME™10 for its leishmanicidal efficacy, tolerability and immunomodulatory activity. METHODS: Normal healthy mice were treated with 3.5 mg/kg single and 7.5 mg/kg single and double doses of KALSOME™10. Liver and kidney function tests were performed fourteen days after treatment. Next, normal mice were infected with Leishmania donovani amastigotes. Two months post infection they were treated with the above mentioned doses of KALSOME™10 and sacrificed one month after treatment for estimation of parasite burden in the liver and spleen by Limiting Dilution Assay. Leishmanial antigen stimulated splenocyte culture supernatants were collected for cytokine detection through ELISA. Flow cytometric studies were performed on normal animals treated with KALSOME™10, Amphotericin B (AmB) and AmBiosome to compare their immunomodulatory activities. RESULTS: The drug was found to induce no hepato- or nephrotoxicities at the studied doses. Moreover, at all doses, it led to significant reduction in parasite burden in two month infected BALB/c mice, with 7.5 mg/kg double dose resulting in almost complete clearance of parasites from both liver and spleen. Interestingly, the drug at 7.5 mg/kg double dose could almost completely inhibit the secretion of disease promoting cytokines, IL-10 and TGFß, and significantly elevate the levels of IFNγ and IL-12, cytokines required for control of the disease. Mice treated with KALSOME™10 showed elevated levels of IFNγ and suppressed IL-10 secretion from both CD4(+) and CD8(+) subsets of T cells, as well as from culture supernatants of splenocytes, compared to that of normal, AmB and AmBisome treated animals. CONCLUSIONS: Treatment of infected mice with 7.5 mg/kg double dose of KALSOME™10 was safe and effective in clearing the parasites from the sites of infection. The drug maintains the inherent immunomodulatory activities of AmB by effectively suppressing disease promoting cytokines IL-10 and TGFß, thereby boosting IL-12 and IFNγ levels. This emphasizes KALSOME™10 as a promising drug alternative for lifelong protection from VL.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis.

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ,under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.

Involvement and interactions of different immune cells and their cytokines in human visceral leishmaniasis

Visceral leishmaniasis (VL) or kala-azar, a disseminated infection of the lymphoreticular system of the body, is marked by severe defect in immune system of the host. Successful cure of VL depends on the immune status of the host in combination with the effects of the antileishmanial drugs. The rationale approach towards eradication of this disease would be to potentiate the immune functioning of the host in addition to parasite killing. This review deals with different aspects of adaptive and innate immune responses and explores their role in protection or pathogenesis of VL. IL-10 has emerged as the principal cytokine responsible for disease pathogenesis, although evidences regarding its source during active VL remain inconclusive. On the other hand, IFNγ, under the influence of IL-12, is mostly correlated with healing of the disease. Chemokines are important in mounting cell-mediated immune response as they can prevent parasite invasion in association with cytokines. Different types of T cells like CD4, CD8 and NK T cells also contribute to the immunology of this disease. In spite of conflicting reports, the role of regulatory T cells in VL pathogenesis is important. Recently discovered Th17 subset and its different members have been reported to perform diverse functions in the course of VL and leishmaniasis as a whole. Innate immune responses, depending on the cell types, are essential in early parasite detection and subsequent development of an efficient NK cell response. Immunotherapy targeting IL-10 could be looked upon as an interesting option for the treatment of VL.

Current diagnosis and treatment of visceral leishmaniasis.

Human visceral leishmaniasis (VL), a potentially fatal disease, is most prevalent in the Indian subcontinent, East Africa and South America. Definite diagnosis and effective treatment are the primary needs for the control of VL. Diagnosis of VL has typically relied on microscopic examination of bone marrow/splenic aspirate, but serology and molecular methods are now better alternatives. The conventional drugs for treatment of VL have limitations including unresponsiveness, relapse, specific toxicities and parenteral administration lasting for long durations. Moreover, they are less effective in HIV-VL-coinfected patients. Registration of miltefosine and paromomycin, and preferential pricing of AmBisome has offered more choices for monotherapy and combination therapy for VL. Combination therapy will increase treatment efficacy and prevent the development of resistance. In addition, active case finding and vector control strategies will also have a positive impact in the control of VL. This article critically addresses the currently available diagnostic and treatment regimens for the control of VL.

A curative immune profile one week after treatment of Indian kala-azar patients predicts success with a short-course liposomal amphotericin B therapy.

BACKGROUND: The present pilot study investigating the minimum dose for short-course single and double-dose treatment of kala-azar with an apparently new liposomal formulation of amphotericin B, Fungisome, led to identification of immunological components for early detection of success and/or failure to cure. METHODS: Patients were treated with 5, 7.5 (single-dose) and 10 mg/kg body weight (5 mg/kg double-dose) of Fungisome. Immunological investigations involving plasma cytokines and antigen-specific lymphoproliferation and cytokine responses from PBMCs were carried out before, 1 week after Fungisome treatment, at the time of relapse, and again after conventional amphotericin B treatment. RESULTS: At 1-month follow-up all the patients showed 100% initial cure. However, total doses of 5, 7.5 and 10 mg/kg Fungisome showed 60%, 50% and 90% cure, respectively, at 6-months posttreatment. Patients successfully cured demonstrated downregulation of IL-12 and IL-10 in plasma, and two-fold or more elevation of IFN-gamma, IL-12 and TNF, and significant down-regulation of IL-10 and TGF-beta in culture supernatants 1-week posttreatment irrespective of drug-dose. A differential immune profile, involving insignificant decline in IL-10 and IL-12 in plasma and negligible elevation of IFN-gamma, IL-12 and TNF, and persistence of IL-10, despite decline in TGF-beta in culture supernatants, in apparently cured individuals, corresponded with relapse within 6-months of treatment. CONCLUSION: Immunological investigations revealed significant curative and non-curative immunomodulation 1-week posttreatment, correlating with successful cure and relapse, respectively. Although immune-correlation was dose-independent, almost consistent curative response in patients treated with the highest dose 10 mg/kg reflected a definitive impact of the higher-dose on the immune response. TRIAL REGISTRATION NAME AND NUMBER: Clinical Trials Registry--India (CTRI) CTRI/2009/091/000764.