Altered T cell subpopulations and serum anti-TYRP2 and tyrosinase antibodies in the acute and chronic phase of alopecia areata in the C3H/HeJ mouse model.

BACKGROUND: C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. OBJECTIVE: We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. METHODS: We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. RESULTS: The CD8+NKG2D+ T and CD8+ TEM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4+ TEM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8+ TEM/TRM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c+ cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8+ T cell percentages were upregulated in AA mice. CONCLUSION: These results suggest that the CD8+ and CD4+ T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

Phenoloxidase is involved in the immune reaction of Helix lucorum to parasitic infestation by dicrocoeliid trematode.

INTRODUCTION: Phenoloxidases are known to play a role in the immune defences of arthropods and molluscs. In the invertebrates, phenoloxidases mediate three major physiologically important processes: sclerotization, wound healing, and defence reactions. Helix lucorum serve as the first intermediate host for the larval stages of dicrocoeliid trematodes which infects animals as well as human beings. OBJECTIVE: The aim of the study is to investigate the effect of larval forms of dicrocoeliid trematodes to phenoloxidase acitivity in H. lucorum, Linneaus, 1758, in Bitlis, Turkey. The effect of the snail's shell colour to phenoloxidase activity was also investigated. MATERIAL AND METHODS: Land snails (n=200) were collected by hand from their natural habitats during the period May - June 2019 in Bitlis, Turkey. Evaluation of the process was performed by measuring immune reaction of the snails against larval forms of dicrocoeliid trematodes. Phenoloxidase activity assay was carried out using a spectrophotometer device based on 3,4-Dihydroxy-L-phenylalanine (L-dopa) hydrolysis. RESULTS: The natural infection rate of the land snails with the developmental stages of dicrocoeliid trematodes was 20%. Phenoloxidase activity was found to be significantly higher (*p<0.05) in larval forms of dicrocoeliid trematodes infected snails when compared with non-infected snails. No effect of shell colours to phenoloxidase activity was observed. CONCLUSIONS: To the best of the authors' knowledge, this study is the first to report that the phenoloxidase system is involved in the immune reaction of Helix lucorum to parasitic infestation by larval forms of dicrocoeliid trematodes.

Interaction of Penaeus vannamei hemocyanin and α2-macroglobulin modulates the phenoloxidase activity.

Prophenoloxidase (proPO)-activating system is a critical innate immune defense in invertebrates. However, the mechanisms involved in regulating the phenoloxidase (PO) activity in shrimp hemolymph remain ill-defined. Our previous studies showed that Penaeus vannamei hemocyanin (HMC) and α2-macroglobulin (α2M), two key regulators of proPO-activating system in plasma, might interact with each other, indicating that this interaction could be implicated in controlling PO activity. Herein, we further confirmed that HMC specifically bind to α2M using Pull down and Far-Western blot analyses. Further studies demonstrated that HMC could directly interact with the receptor binding domain of α2M. In addition, HMC and α2M followed similar expression pattern upon Vibrio parahaemolyticus infection, suggesting the interaction of HMC and α2M might have a role in immune response. Finally, we found that α2M, as a broad-spectrum proteinase inhibitor, suppressed the serum PO activity in vitro, while hemocyanin could partially restore this inhibitory effect. In sum, the present data indicate that HMC interacts with α2M and therefore modulates the PO activity. This finding contributes to better understanding of stable state maintenance of PO activity in shrimp.

Multifunctional role of a fungal pathogen-secreted laccase 2 in evasion of insect immune defense.

Laccases are widely present in bacteria, fungi, plants and invertebrates and involved in a variety of physiological functions. Here, we report that Beauveria bassiana, an economic important entomopathogenic fungus, secretes a laccase 2 (BbLac2) during infection that detoxifies insect immune response-generated reactive oxygen species (ROS) and interferes with host immune phenoloxidase (PO) activation. BbLac2 is expressed in fungal cells during proliferation in the insect haemocoel and can be found to distribute on the surface of haemolymph-derived in vivo fungal hyphal bodies or be secreted. Targeted gene-knockout of BbLac2 increased fungal sensitivity to oxidative stress, decreased virulence to insect, and increased host PO activity. Strains overexpressing BbLac2 showed increased virulence, with reduced host PO activity and lowered ROS levels in infected insects. In vitro assays revealed that BbLac2 could eliminate ROS and oxidize PO substrates (phenols), verifying the enzymatic functioning of the protein in detoxification of cytotoxic ROS and interference with the PO cascade. Moreover, BbLac2 acted as a cell surface protein that masked pathogen associated molecular patterns (PAMPs), enabling the pathogen to evade immune recognition. Our data suggest a multifunctional role for fungal pathogen-secreted laccase 2 in evasion of insect immune defenses.

Expression of a phenoloxidase cascade inhibitor enhances the virulence of the fungus Beauveria bassiana against the insect Helicoverpa armigera.

Entomopathogenic fungi have high potential for controlling insect pests, although the slow killing speed has blocked their widespread application. To increase the virulence of entomopathogenic fungi, genetic modification can be employed. Egf1.0 is an immunosuppressive protein encoded by polydnavirus, carried by parasitoid wasp Microplitis demolitor, which blocks the prophenoloxidase (PPO) activation response of host insects. In this study, we explored the feasibility of genetically modifying entomopathogenic fungi with increased virulence by expressing Egf1.0. In comparison with the wild-type parents, the median lethal concentration (LC50) of Beauveria bassiana expressing Egf1.0 against Helicoverpa armigera was reduced by 2.7-fold, and the median lethal time (LT50) was reduced by 22.8%. In vitro assay showed that recombinant Egf1.0 was able to inhibit the PPO activation response of H. armigera. In vivo assay revealed that the expression of Egf1.0 in B. bassiana caused a higher degree of suppression to PPO activation response of H. armigera. These assays suggested that the increased virulence of the transgenic fungi is due to the increased ability to suppress the host insect's immune response. Moreover, colony growth, conidia yield, and germination assays revealed that the expression of Egf1.0 in B. bassiana had no effect on its growth and development. In conclusion, the expression of Egf1.0 can significantly enhance the pathogenicity of B. bassiana against host insects.

Autoimmune response against tyrosinase induces depigmentation in C57BL/6 black mice.

Regulation of melanogenesis by tyrosinase has now become an attractive approach for treatment of vitiligo but still the role of tyrosinase in the induction of depigmentation remains largely unexplored. This study was explored the role of tyrosinase in the induction of autoimmune depigmentation in C57BL/6 mice. Depigmentation was induced in C57BL/6 mice by tyrosinase immunization. Induced depigmentation was characterized by visual detection and was verified by histopathological analysis of lesional and non-lesinal skin biopsies. Moreover, induced depigmentation was re-validated by gene expression analysis of vitiligo-relevant genes by Taqman assays. Immunization of C57BL/6 mice by tyrosinase induces depigmentation on hairs as well as on skin. Immunoassays with Protein A-purified immune IgGs showed high titre antibodies against tyrosinase. Histopathological analysis showed that the total melanocytes were depleted from the basal layer of the epidermis and also from the dermis of depigmented lesions. The gene expression of vitiligo-relevant genes TYRP1, DCT, MLANA, MCIR, POMC, FOXJ2, CSNK1G3, SOX10, PMEL and KIT was significantly low in lesional skin as compared with non-lesional skin (p < .05). In contrast, the mRNA expression of CASP3 and NFκB1 was significantly high in lesional skin of depigmented mice as compared with non-lesional skin (p < .05). Furthermore, involvement of cellular immunity in depigmentation was confirmed by the reduction of CD4+:CD8+ lymphocytes ratio. In conclusion, this study shows that the autoimmune response against tyrosinase induces depigmentation in black C57BL/6 mice. The data obtained from the lesional and non-lesional skin biopsies showed the same features as were reported in human vitiligo patients.

Ciona robusta hemocyte populational dynamics and PO-dependent cytotoxic activity.

Hemocyte populations from the ascidian Ciona robusta, separated through a Percoll discontinuous density gradient, are further characterized by May-Grünwald-Giemsa staining and a cytochemical reaction for phenoloxidase. Variability in cell density, acidophilic property and phenoloxidase activity suggest multiple hemocyte type populations, cell lineages and morphotypes that may be involved in distinct cellular responses. Therefore, unilocular refractile granulocytes, typical of this ascidian species, enriched in a fraction separated from the hemolymph show in vitro phenoloxidase-dependent cytotoxic activity against mammalian erythrocytes and a tumor cell lineage, in addition the properties listed above indicate relationships with vacuolated signet ring cells. Finally, bromo-deoxyuridine with, diamino-phenylindole fluorescent reaction and May-Grünwald-Giemsa staining show that in the hemolymph there are hyaline amoebocytes and granulocytes with potential proliferating activity. Present findings and reviewed images of previously reported inflammatory hemocytes in the tunic and pharynx allow us to speculate on theoretical outlines of hemocyte differentiation pathways.

Effect of thermal stress on the immune responses of Chilo suppressalis walker (Lepidoptera: Crambidae) to Beauveria bassiana.

Temperature is one of the important environmental elements affecting ecological fitness of insects through alterations in physiological systems. In the current study, a comparison was made on the cellular and humoral immune responses of the Chilo suppressalis larvae exposed to thermal stress (34 °C) and optimal rearing temperature (24 °C). Although total hemocyte count increased in the injected larvae by Beauveria bassiana, elevation of hemocyte numbers was significantly different in the larvae exposed to 34 °C for a short-time period compared to long-term exposure and control. A similar trend was observed in plasmatocyte and granulocyte counts as well as phenoloxidase activity. Gene expression of some antimicrobial peptides, including attacin1, attacin2, cecropin1, cecropin2, defensin, gallerimycin, lysozyme and prophenoloxidase-activating proteinase-3, was compared in the larvae exposed to thermal regimes and injection challenges. In all cases, expression of the target genes was relatively higher in the larvae injected by B. bassiana and short-term exposure at 34 °C. The present results confirmed that C. suppressalis could modulate the immune system in response to different thermal stress conditions mainly over a short period.

Characterization and expression of tyrosinase-like genes in the anemone Exaiptasia pallida as a function of health and symbiotic state.

Coral disease is a major threat to reef ecosystems and therefore, understanding the cellular pathways underlying disease progression and resistance is critical to mitigating future outbreaks. This study focused on tyrosinase-like proteins in cnidarians, which contribute to melanin synthesis, an invertebrate innate immune defense. Specifically, characterization and phylogenetic analysis of cnidarian tyrosinases were performed, and their role in symbiosis and a "mystery disease" in the anemone Exaiptasia pallida was investigated using qPCR. The results reveal a diversity of tyrosinase-like proteins in cnidarians that separate into two major clades on a phylogenetic tree, suggesting functional divergence. Two E. pallida sequences, Ep_Tyr1 and Ep_Tyr2, were further investigated, and qPCR results revealed no gene expression differences as a function of symbiotic state, but decreased expression in late disease stages. Overall this work provides evidence for the participation of tyrosinases in the cnidarian immune response.

Functional conservation and division of two single-carbohydrate-recognition domain C-type lectins from the nipa palm hispid beetle Octodonta nipae (Maulik).

As an invasive pest, the complete and effective innate immune system is crucial for the nipa palm hispid beetle Octodonta nipae (Maulik) to adjust to new environments. C-type lectins (CTLs) are large families of carbohydrate-binding proteins that possess one or more characteristic carbohydrate-recognition domains (CRD) and function as pattern-recognition receptors, which play important roles in mediating humoral and cellular immunity. In the present study, for the first time, we report two CTL-Ss (single-CRD CTLs) from O. nipae (Maulik) (designated OnCTL1 and OnCTL2). The two CTL-Ss share high identity at conserved amino acids associated with conserved carbohydrate binding sites Gln-Pro-Asp (QPD) motifs and clearly show a 1:1 orthologous relationship in insects, which endow them with functional conservation and diversification. mRNA abundance analysis showed that OnCTL1 was upregulated upon Staphylococcus aureus and Escherichia coli challenge at 6 and 12 h, while OnCTL2 underwent no changes upon E. coli challenge and was even downregulated after S. aureus infection. Knockdown of OnCTL1 significantly decreased the transcripts of two key serine proteases (prophenoloxidase activating factors), OnPPAF1 and OnPPAF3, followed by the reduction of haemolymph phenoloxidase activity; it also increased the expression of Defensin2B. In contrast, silencing of OnCTL2 significantly decreased the expression of Defensin2B and Attacin3C, the encapsulation index, and the phagocytosis rate compared to the dsEGFP group. The spreading results showed that more irregularly shaped plasmatocytes and lower levels of aggregation were found in OnCTL2-silenced pupae than in the dsOnCTL1 and dsEGFP groups. We can infer from the results of this study that the two OnCTLs play important roles in the immune system and generate a functional division: OnCTL1 seems to function more in humoral immunity including mediating bacterial recognition and activating the phenoloxidase cascade, and OnCTL2 plays a greater role in enhancing cellular immunity. These observations could replenish information on the functional diversification of insect CTLs, and also provide valuable information to unravel the immunity in O. nipae.

Engineering antigen-specific NK cell lines against the melanoma-associated antigen tyrosinase via TCR gene transfer.

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T-lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL-TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off-target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/ß chains, along with CD3 subunits, enables the functional expression of an antigen-specific TCR complex on NK cell lines NK-92 and YTS, demonstrated by using a TCR against the HLA-A2-restricted tyrosinase-derived melanoma epitope, Tyr368-377 . Most importantly, the introduction of a TCR complex to NK cell lines enables MHC-restricted, antigen-specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of "mispairing" in TCR gene therapy.

Natural T cell autoreactivity to melanoma antigens: clonally expanded melanoma-antigen specific CD8 + memory T cells can be detected in healthy humans.

We used four-color ImmunoSpot® assays, in conjunction with peptide pools that cover the sequence of tyrosinase (Tyr), melanoma-associated antigen A3 (MAGE-A3), melanocyte antigen/melanoma antigen recognized by T cells 1 (Melan-A/MART-1), glycoprotein 100 (gp100), and New York esophageal squamous cell carcinoma-1 (NY-ESO-1) to characterize the melanoma antigen (MA)-specific CD8 + cell repertoire in PBMC of 40 healthy human donors (HD). Tyr triggered interferon gamma (IFN-γ)-secreting CD8 + T cells in 25% of HD within 24 h of antigen stimulation ex vivo. MAGE-A3, Melan-A/MART-1, and gp100 also induced recall responses in 10%, 7.5%, and 2.5% of HD, respectively. At this time point, these CD8 + T cells did not yet produce GzB (granzyme B). However, they engaged in GzB production after 72 h of antigen stimulation. By this 72-h time point, 57.5% of the HD responded to at least one, and typically several, of the MA. A closer characterization of the Tyr-specific CD8 + T cell repertoire indicated that it was low-affinity, and to primarily entail a stem cell-like subpopulation. Collectively, our data reveal pre-existing endogenous T cell immunity against melanoma antigens in healthy donors, and analogous to natural autoantibodies, we have termed this "natural T cell autoreactivity".

Manduca sexta hemolymph protease-2 (HP2) activated by HP14 generates prophenoloxidase-activating protease-2 (PAP2) in wandering larvae and pupae.

Melanization is a universal defense mechanism of insects against microbial infection. During this response, phenoloxidase (PO) is activated from its precursor by prophenoloxidase activating protease (PAP), the terminal enzyme of a serine protease (SP) cascade. In the tobacco hornworm Manduca sexta, hemolymph protease-14 (HP14) is autoactivated from proHP14 to initiate the protease cascade after host proteins recognize invading pathogens. HP14, HP21, proHP1*, HP6, HP8, PAP1-3, and non-catalytic serine protease homologs (SPH1 and SPH2) constitute a portion of the extracellular SP-SPH system to mediate melanization and other immune responses. Here we report the expression, purification, and functional characterization of M. sexta HP2. The HP2 precursor is synthesized in hemocytes, fat body, integument, nerve and trachea. Its mRNA level is low in fat body of 5th instar larvae before wandering stage; abundance of the protein in hemolymph displays a similar pattern. HP2 exists as an active enzyme in plasma of the wandering larvae and pupae in the absence of an infection. HP14 cleaves proHP2 to yield active HP2. After incubating active HP2 with larval hemolymph, we detected higher levels of PO activity, i.e. an enhancement of proPO activation. HP2 cleaved proPAP2 (but not proPAP3 or proPAP1) to yield active PAP2, responsible for a major increase in IEARpNA hydrolysis. PAP2 activates proPOs in the presence of a cofactor of SPH1 and SPH2. In summary, we have identified a new member of the proPO activation system and reconstituted a pathway of HP14-HP2-PAP2-PO. Since high levels of HP2 mRNA were present in integument and active HP2 in plasma of wandering larvae, HP2 likely plays a role in cuticle melanization during pupation and protects host from microbial infection in a soil environment.

In the ovary of Ciona intestinalis (Type A), immune-related galectin and phenoloxidase genes are differentially expressed by the follicle accessory cells.

Riboprobes (in situ hybridization) and antibodies (immunohistochemistry), previously used to show the upregulation of Ciona intestinalis (Type A) galectins (CiLgals-a, CiLgals-b) and phenoloxidase (CinPO2) immune-related genes, were tested on histological sections of the ovary. The ovarian follicles are composed of oocytes encased by follicular cells (FCs) and test cells (TCs). Results show the transcription upregulation of both CiLgals and CinPO2 genes in the vitellogenic FCs, conversely distinct cytolocalization of the proteins are shown. At vitellogenic stage, the CiLgals are localized in the FCs, in the oocyte cytoplasm, and close to the germinal vesicle (GV), whereas the CinPO2 was never identified in the FCs. In a presumptive advanced phase and at the post-vitellogenic stage the TCs appear to be labelled by the CinPO2 riboprobe, and the protein identified by the antibody suggesting an mRNA transcytosis process from FCs. At post-vitellogenic stage the CiLgals mainly enrich the GV nucleoplasm, whereas the CinPO2 is contained in TCs and in the ooplasm but never found in the GV. This finding sheds new light on a former paper in which TCs were reported to be the only CinPO2-producing cells in the ovarian follicle. Finally, CiLgals and CinPO2 genes transcription and proteins production seem to be associated with accessory cells during their differentiation from vitellogenic to post-vitellogenic stage. The present findings promote further research on the early upregulation of immune-related genes, and the potential multifunctional role of the produced proteins. In addition further insight on the accessory cells involvement in ascidian oogenesis are reported.

Comparative study of three phenoloxidases in the sea cucumber Apostichopus japonicus.

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus, the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3'- and 5'-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052, named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs.

Variation in the immune state of Gammarus pulex (Crustacea, Amphipoda) according to temperature: Are extreme temperatures a stress?

Temperature is known to impact host-parasite interactions in various ways. Such effects are often regarded as the consequence of the increased metabolism of parasites with increasing temperature. However, the effect of temperature on hosts' immune system could also be a determinant. Here we assessed the influence of temperature on the immunocompetence of the crustacean amphipod Gammarus pulex. Amphipods play a key ecological role in freshwater ecosystems that can be altered by several parasites. We investigated the consequences of three weeks of acclimatization at four temperatures (from 9 °C to 17 °C) on different immunological parameters. Temperature influenced both hemocyte concentration and active phenoloxidase enzymatic activity, with lower values at intermediate temperatures, while total phenoloxidase activity was not affected. In addition, the ability of gammarids to clear a bacterial infection was at the highest at intermediate temperatures. These results suggest a dysregulation of the immune system of gammarids in response to stress induced by extreme temperature.

Early-life inflammation, immune response and ageing.

Age-related diseases are often attributed to immunopathology, which results in self-damage caused by an inappropriate inflammatory response. Immunopathology associated with early-life inflammation also appears to cause faster ageing, although we lack direct experimental evidence for this association. To understand the interactions between ageing, inflammation and immunopathology, we used the mealworm beetle Tenebrio molitor as a study organism. We hypothesized that phenoloxidase, an important immune effector in insect defence, may impose substantial immunopathological costs by causing tissue damage to Malpighian tubules (MTs; functionally equivalent to the human kidney), in turn accelerating ageing. In support of this hypothesis, we found that RNAi knockdown of phenoloxidase (PO) transcripts in young adults possibly reduced inflammation-induced autoreactive tissue damage to MTs, and increased adult lifespan. Our work thus suggests a causative link between immunopathological costs of early-life inflammation and faster ageing. We also reasoned that if natural selection weakens with age, older individuals should display increased immunopathological costs associated with an immune response. Indeed, we found that while old infected individuals cleared infection faster than young individuals, possibly they also displayed exacerbated immunopathological costs (larger decline in MT function) and higher post-infection mortality. RNAi-mediated knockdown of PO response partially rescued MTs function in older beetles and resulted in increased lifespan after infection. Taken together, our data are consistent with a direct role of immunopathological consequences of immune response during ageing in insects. Our work is also the first report that highlights the pervasive role of tissue damage under diverse contexts of ageing and immune response.

Immunomodulatory effects of temperature and pH of water in an Indian freshwater sponge.

Eunapius carteri, a freshwater sponge of India, inhabits the ponds and lakes and experiences variations of temperature and pH of water throughout the year. Sponges bear evolutionary and ecological importance with limited information on their immunological attribute and adaptational resilience in a changing environment. This paper reports temperature and pH specific responses of immune related parameters in sponge maintained in the experimental conditions of laboratory. Innate immunological parameters like phagocytosis and generation of cytotoxic molecules like superoxide anion, nitric oxide and phenoloxidase activity were estimated in E. carteri at different environmentally realistic water temperatures (10, 20, 30 and 40°C) and pH (6.4, 7.4 and 8.4). Phagocytosis and cytotoxicity are established as important immune parameters of invertebrates. Calalase, an antioxidant enzyme and phosphatases are involved in pathogen destruction and are considered as components of innate immunity. Activities of catalase, acid and alkaline phosphatases were estimated in E. carteri at different thermal regimes and pH. Modulation of phagocytic and cytotoxic responses and the activities of catalase and phosphatases at different water temperatures and pH indicated temperature and pH specific immunological status of E. carteri. Present investigation deals with the effects of selected hydrological parameters on the fundamental immune related parameters in sponge indicating its adaptational plasticity. Immunological resilience of this species in the face of variation of water temperature and pH is thought to be a special adaptive feature of sponge, a reported "living fossil".