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• Front Immunol
• PMC3342266

Front Immunol. 2012; 3: 94.

P3 mAb: An Immunogenic Anti-NeuGcGM3 Antibody with Unusual Immunoregulatory Backdrop

Darel Martínez

^oneTumor Immunology Direction, Eye of Molecular Immunology, Havana, Cuba

Nely Rodríguez

^oneTumor Immunology Direction, Heart of Molecular Immunology, Havana, Cuba

Tania Griñán

¹Tumor Immunology Management, Heart of Molecular Immunology, Havana, Cuba

Teresa Rondón

¹Tumor Immunology Management, Eye of Molecular Immunology, Havana, Cuba

Ana María Vázquez

^twoInnovation Management Direction, Eye of Molecular Immunology, Havana, Cuba

Rolando Pérez

³Molecular Immunology Institute, Center of Molecular Immunology, Havana, Republic of cuba

Ana María Hernández

¹Tumor Immunology Management, Centre of Molecular Immunology, Havana, Cuba

Received 2011 Nov 25; Accepted 2012 Apr 11.

Abstract

P3 is a murine IgM mAb that recognize North-glycosylated gangliosides, sulfatides, and antigens expressed in melanoma, breast, and lung human tumors. This antibody has the power to trigger an IgG antibody response in the syngeneic BALB/c model, even when it is administered in the absenteeism of adjuvant or carrier protein. The mechanism past which the P3 mAb, a self-immunoglobulin, induce this immune response in the absenteeism of co-stimulatory or classical danger signals is still unknown. In the present paper nosotros show that the high immunogenicity of P3 mAb depends not simply on CD4 but too on CD8⁺ T cells, since the depletion of CD8⁺ or CD4⁺ T cells led to the loss of P3 mAb immunogenicity in the syngeneic model. Furthermore, the immunization with P3 mAb enhanced the recovery of the CD8⁺ T cell population in mice treated with an anti-CD8a antibody. Additionally, the immunization with P3 mAb restored the capacity of immunosuppressed mice to pass up allogeneic tumors, a mechanism mediated past the activeness of CD8⁺ T cells. Finally, in mice with cyclophosphamide induced lymphopenia, the assistants of P3 mAb accelerated the recovery of both CD4⁺ and CD8⁺ T cells. These results show new possibilities for B and CD8⁺ T cells interactions during the immune response elicited by a self-protein. Furthermore they signal to P3 mAb as a potential interesting candidate for the treatment of immunosuppressed patients.

Keywords: CD8 T cells, immunogenicity, immunoregulation, monoclonal antibody

Introduction

P3 mAb is an Ab1 antibody that recognizes NeuGc-containing gangliosides, sulfated glycolipids, and antigens present in different human tumors including those from the lung, breast, and melanoma (Vazquez et al., 1995). VH P3 is of germ line origin and belongs to the Q52 (VH II) gene family, previously observed in autoantibodies against gangliosides and oftentimes used by CD5⁺ B lymphocytes (Perez et al., 2001). This antibody is capable of triggering a stiff anti-idiotypic (Ab2) response in the syngeneic model, fifty-fifty in the absence of adjuvant or carrier poly peptide (Vazquez et al., 1998), which is a miracle rarely observed (Baskin et al., 1990; Maruyama et al., 2002).

Some authors have suggested that the IgM isotype confers sure advantages for the development of anti-idiotypic responses (Baskin et al., 1990; Reitan and Hannestad, 1995, 2002), but it has been shown that this is not a general property of the IgM anti-ganglioside antibodies (Vazquez et al., 1998; Reitan and Hannestad, 2001), which suggest that the variables regions of the immunogenic antibodies could contain antigenic determinants that could exist responsible for their unusual immunogenicity. In fact, the immunogenicity of P3 mAb idiotype has too been demonstrated in the absence of any constant domain (Rodriguez et al., 2007). Some authors have shown that the presence of somatic hypermutations is an important factor in the immunogenicity of autologous immunoglobulins (Zaghouani et al., 1992; Eyerman and Wysocki, 1994; Cao et al., 1995; Eyerman et al., 1996; Wysocki et al., 1998; Zhang et al., 2001). However, P3 mAb variable region is coded by germ line genes (Perez et al., 2001), so the presence of somatic hypermutations is non the cause of its high immunogenicity (Rodriguez et al., 2007). In fact, Reitan and Hannestad (2001) analyzed the syngeneic immunogenicity, in the absence of adjuvant, of the idiotypes of 73 mAb of IgM isotype. These researchers reported that the 4 antibodies that generated high levels of anti-idiotypic IgG antibodies, were coded by germ line genes.

Based on structural modeling and immunogenetic analysis, several P3 mAb mutants were obtained by substituting the basic residues R31, R98, and R100a, of the H-CDRs of the variable regions of P3. Immunization experiments with these mutants proved that these basic residues play a critical role in both its binding specificity and high immunogenicity (Lopez-Requena et al., 2007a,b).

The fact that P3 mAb induces anti-idiotypic antibodies of IgG isotype suggests the participation of T cells in the generation of this response. This is very interesting since P3 is a cocky-immunoglobulin, presented to the immune system in the absence of the classical co-stimulatory signals (Caux et al., 1994; Tseng et al., 2001). Previous data already published by our group showed that lymph node cells from BALB/c mice immunized with P3 mAb proliferated in vitro, in a dose-dependent manner, not but in the presence of P3 mAb but also in the presence of 1E10, one of its anti-idiotypic Ab2 mAb. This result suggests that P3 mAb is able to actuate B–T cell idiotypic networks (Perez et al., 2002).

All these results prompted us to evaluate the immunogenicity of P3 mAb in unlike mouse strains, and to written report the importance of different T cell populations in the consecration of this unusual anti-idiotypic response, which could contribute to understand the mechanism underlying this unusual immunogenicity. Although for many years it was believed that the Th1/Th2 paradigm only practical to CD4⁺ T cells, several studies have shown that CD8⁺ T cells can too provide B prison cell help (Jacobsen et al., 1993; Cronin et al., 1995; Hermann et al., 1995). Thus in the nowadays work we wanted to written report the contribution of both CD4⁺ and also CD8⁺ T cells in the induction of an anti-idiotypic response.

Results

Humoral response to P3 mAb

Iii important factors to narrate the immune response against a given antigen are the kinetic of the response, the influence of the immunization route, and the importance of the employ of an adjuvant to achieve the response. To assess the significance of these factors for P3 mAb immunogenicity, outset, BALB/c mice were immunized intraperitoneally, intramuscularly, or subcutaneously with four doses of this antibody in phosphate buffered saline (PBS). We detected an antibiotic response against P3 mAb in virtually immunized animals regardless the road of immunization used. All the same the level of the antibody response was significantly college in animals immunized subcutaneously (Figure ​ 1).

IgG antibodies response in BALB/c mice immunized with P3 mAb using different routes of immunization. BALB/c mice were immunized with 4 doses of P3 mAb in PBS using dissimilar routes of immunization: subcutaneously (s.c.), intraperitoneally (i.p.), or intramuscular (i.one thousand.). The blood samples were obtained before the immunization and seven days after the second, tertiary, and fourth doses (days 21, 35, and 49 of the immunization protocol). Sera were diluted one:100 and the response against purified P3 mAb was assessed by ELISA, reactivity was adamant using element of group i phosphatase-conjugated goat anti-mouse IgG (Fcγ-specific). Values are expressed as means ± SD (n = 5). *p < 0.05, Mann–Whitney U test, one-tailed.

Next we studied if the presence of an adjuvant changes P3 immunogenicity. For this purpose we evaluated the IgG antibiotic response induced in BALB/c mice immunized subcutaneously with P3 only in PBS or emulsified in Freund's adjuvant. Furthermore we compare the response induced in mice that received four doses of P3 mAb at ii weeks intervals or a single dose of the antibody. Every bit is shown in Figure ​ 2, the mice immunized with four doses of P3 mAb generated an antibody response of the IgG isotype independently on the apply of Freund's adjuvant. This response was specific for P3 mAb idiotype since did not cross react with an irrelevant IgM used as isotype control (Figure ​ A1 in Appendix). The response was detected from the 2nd dose for the animals immunized with adjuvant and from the third for those immunized only with P3/PBS. Furthermore, mice immunized with a single dose of P3 mAb in the presence of Freund'south adjuvant, elicited an IgG antibiotic response detectable at 24-hour interval 21 after immunization, moment equivalent to the second dose of the mice that received four doses. Although these mice received only one dose of P3 mAb, the anti-P3 antibody response connected increasing in time and 49 days afterwards immunization reached levels that did not show significant differences with those obtained in the animals immunized with four doses. Nosotros could non observe any antibody response of the IgG isotype in the animals that received just one dose of P3 mAb in PBS (Figure ​ ii).

Kinetic of IgG antibodies response in BALB/c mice. Mice were immunized with i (1d) or 4 (4d) doses of P3 mAb in PBS or emulsified in Freund's adjuvant (FA). The blood samples were obtained before the immunization and on days 7, 21, 35, and 49 of the immunization protocol. Sera were diluted 1:100 and the reactivity against purified P3 mAb was assessed by ELISA. Binding was adamant using alkaline phosphatase-conjugated caprine animal anti-mouse IgG (Fcγ-specific). Values are expressed as ways ± SD (n = 5).

Since P3 mAb has the rare ability to generate humoral immune response in the syngeneic BALB/c model, we wanted to establish whether it was as immunogenic in a different mouse strain, like C57BL/six. The Figure ​ 3 showed that, in dissimilarity with the results obtained in syngeneic BALB/c mice (Figure ​ 3A), in the allogeneic inbred mice strain C57BL/half dozen, after four doses of P3 mAb immunization we could not detected whatever meaning IgG antibiotic response at the dilution used (Figure ​ 3B). Nonetheless in the outbred NMRI mice strain we could observe a pregnant IgG response afterward four doses in all the mice tested (Figure ​ threeC).

Immunogenicity of P3 mAb in different mice strains. BALB/c (A), C57BL/vi (B), or NMRI (C) mice were immunized subcutaneously with four doses of P3 mAb in PBS every 2 weeks. The sera samples were taken before the immunization period (day 0) and afterward the third (day 35) and quaternary (solar day 49) dose. The reactivity was determined by ELISA in P3 mAb coated plates using element of group i phosphatase-conjugated caprine animal anti-mouse IgG (Fcγ-specific). For each mouse, is graphed the means ± SD of each of the values obtained in triplicate.

Importance of T cells in the antibiotic response confronting P3 mAb

Since the immunization with P3 mAb induce antibodies of IgG isotype and depends on the mouse strain, next we evaluated the participation of T cells in the consecration of this antibody response. For this purpose CD4⁺ or CD8⁺ T cells were depleted with anti-CD4 or anti-CD8a antibodies at different moments during the immunization protocol and the antibody response after the fourth dose was tested past ELISA. As is shown in Effigy ​ iv, a meaning decrease in the antibody response was detected in the sera of the animals treated earlier the first dose of P3 mAb (group 2), with the anti-CD4 (Figure ​ 4A) but besides in those treated with the anti-CD8a antibody (Figure ​ 4B). There were non significant differences in the response of the groups treated before the second and the 3rd dose with any of the depleting antibodies (grouping iii and 4; Figure ​ fourC).

Differences in the IgG antibodies response against P3 mAb in BALB/c mice treated in different moments of the protocol with CD4a or CD8a depleting antibodies. BALB/c mice were immunized with four doses of P3 mAb in PBS and were treatment with an anti-CD4a (A) or anti-CD8a (B) depleting antibiotic only once before 1 of the dose of P3 (except the quaternary). Sera were diluted one:100 and the reactivity against purified P3 mAb was assessed by ELISA. Binding was determined using alkaline phosphatase-conjugated caprine animal anti-mouse IgG (Fcγ-specific). (C) A representative effect on CD8⁺ cell populations of the handling with i dose of anti-CD8a depleting antibody, upper figure showed command mouse and lower figure showed treated mouse. Values are expressed as ways ± SD (n = 5). *p < 0.05, Isle of mann–Whitney U exam, one-tailed.

In vivo restoration of CD8⁺T lymphocytes past P3 mAb

Due to the involvement of CD8⁺ T cells in the induction of the antibody response confronting P3 mAb, we studied if the immunization with this antibody increase the pct of CD8⁺ T cells in the inguinal lymph node of BALB/c mice treated with the anti-CD8a antibody. The results showed that the percentage of CD8⁺ T cells in mice immunized with P3 mAb was significantly higher than the one detected in mice treated with the command antibody or PBS (Figure ​ 5A). In the C57BL/6 strain, where P3 is not immunogenic, we could not find whatsoever deviation in the CD8⁺ T cells percentage between the mice immunize with P3 or with the controls (Figure ​ 5B).

Regeneration of the population of CD8⁺ T cells past the action of the P3 mAb. BALB/c (A) or C57BL/6 (B) mice inoculated with P3 mAb, control mAb (E1), or PBS were intraperitoneally treated with anti-CD8a depleting antibiotic. The expression of the CD8a molecule in lymph node cells was determined past flow cytometry for which the cells were incubated with a fluorescein isothiocyanate-conjugated anti-mouse CD8a. Values represent ways ± SD (n = viii). *p < 0.05, Dunnett t-exam, one-tailed.

Upshot of P3 mAb in F3II mediated engraftment of allogeneic B16-F10 tumor in BALB/c mice

Side by side nosotros tested the ability of this antibody to stimulate an immune arrangement affected for the presence of a tumor. We used a model of tumor induced immunosuppression where BALB/c mice were inoculated with the syngeneic F3II tumor cells, which make them susceptible to the engraftment of the allogeneic B16-F10 tumor cells. One week before the syngeneic tumor inoculation the mice received the kickoff of four, biweekly, doses of P3 mAb, and the allogeneic tumor evolution on immunized and not treated mice was compared. As shown in Figure ​ 6, the immunization with P3 mAb increased the percentage of animals able to reject the allogeneic tumor. L percent of non-immunized mice developed allogeneic B16-F10 in comparing with only xix% of P3 mAb immunized mice. P3 mAb did not have any effect on the kinetics of engraftment or the tumor size in the mice that did not reject the allogeneic tumor (data not shown).

Immunization with P3 mAb retrieves the ability of allogeneic MB16F10 tumor rejection in BALB/c mice immunosuppressed by syngeneic F3II tumor. Mice inoculated with F3II tumor and treated or not with P3 mAb were challenged with MB16F10 tumor. The bars indicate the percentage of mice developing tumors. The values represent the mean of 2 dissimilar experiments. Ctrl-: BALB/c mice that were not inoculated with F3II tumor, Ctrl+: C57BL/6 mice, in which the tumor MB16F10 are syngeneic. *p < 0.05, Pearson'due south chi-square (χ²) test, i-tailed.

Capacity of P3 mAb to recovery T jail cell populations in lymphopenic mice

To farther evaluate the therapeutic potential of this antibody, we tested the ability of P3 mAb to stimulate in vivo the recovery of unlike lymphocyte populations in a model of induced lymphopenia (Zuluaga et al., 2006) that resemble the state of immune suppressed patients. BALB/c mice treated with cyclophosphamide were inoculated with P3 or an irrelevant mAb, and the recovery of the lymphocyte populations was measured past flow cytometry. As is shown in Figure ​ 7, mice that received P3 mAb and not the ones treated with the control mAb, showed a pregnant increase in the percentage of CD4⁺ and CD8⁺ T populations later on the cyclophosphamide treatment. Mice that received just cyclophosphamide or cyclophosphamide plus control mAb showed 18–21% of CD4⁺ T cells and 14–19% of CD8⁺ T cells regarding the normal cell number in not immune-compromised mice. However P3 treated mice showed an increase upward to 46% for CD4⁺ T and fifty% for CD8⁺ T cells. There were no significant differences in the B cells percentages between the dissimilar groups (Effigy ​ 7). This result is not observed in C57BL/6 mice where we could non detect differences betwixt the controls and the mice treated with P3 mAb (data non shown).

Recovery of the populations of CD4⁺ and CD8⁺ T cells by immunization with P3 mAb in BALB/c mice treated with cyclophosphamide. Mice were inoculated with two doses of cyclophosphamide and then received an intravenous dose of the P3 mAb or control mAb (E1). It was adamant the amount of cells in inguinal lymph nodes (A), and subpopulations of CD4⁺ T (B), CD8⁺ T (C), and B cells (D) by flow cytometry. It is considered as 100% the number of cells from animals not treated with cyclophosphamide (PBS group). Values represent means ± SD (n = five) in a representative experiment of three. *p < 0.05, Dunnett t-test, i-tailed.

Give-and-take

The high immunogenicity of autologous immunoglobulins is non a miracle frequently observed (Ismaili et al., 1995), and in item the generation of a response confronting anti-ganglioside mAb in the syngeneic model has been reported in very few studies, always using adjuvant or carrier protein (Chapman and Houghton, 1991; Saleh et al., 1993). All the same, P3 mAb is a germline anti-NeuGcGM3 IgM that is highly immunogenic, in the absence of adjuvant or carrier protein, in the syngeneic model. Furthermore, the immunization with this antibiotic induces non just B cell, simply also T cell idiotypic networks (Perez et al., 2002). The first aim of our work was to further written report the high immunogenicity of this antibody and to evaluate the function of T cells in this miracle.

At that place have been reports about variations in the consecration of humoral responses confronting a given antigen depending on the route of inoculation used (Fynan et al., 1993; Raz et al., 1994). Although we detected an antibiotic response against P3 mAb when it was inoculated by subcutaneous, intramuscular, or intraperitoneal road, this response was significantly higher past the subcutaneous immunization. This could be related with a slower speed of antigen release or due to differences in the specific APC populations in the site of immunization.

The generation of an antibody response against a proteic antigen, such as antibodies, is mostly favored by the number of doses and by the presence of adjuvant (Cox and Coulter, 1997; Stills, 2005). When P3 mAb was inoculated merely in PBS, at least iii doses were necessary to induce the allowed response. However when it was inoculated in the presence of Freund's adjuvant, simply i dose was enough to induce an IgG response that had not significance differences, regarding the magnitude or the kinetic, with the one achieved with four doses. This result confirms the ability of this antibody to exist autoimmunogenic in the syngeneic model and suggest that the tedious release of small amounts of the antibody to the circulation, induced by the depot event of the adjuvant, favor the consecration of a strong allowed response. On the other hand this could be a result of the capacity of this antibody to activate T cell idiotypic networks (Perez et al., 2002; Rodriguez et al., 2007).

Evidence that suggest the participation of T cells in the anti-P3 response is the fact that despite the high immunogenicity in BALB/c mice, at that place was no antibody response against P3 mAb after the immunization of C57BL/6 mice with four doses of the mAb in PBS. These differences in the humoral response against P3 mAb betwixt in dissimilar mice strains could be because T cells recognize P3 mAb epitopes presented past BALB/c strain specific MHC haplotypes, as has been described for other antigens (Misharin et al., 2009). On the other paw, P3 mAb likewise induced an anti-idiotypic response in NMRI mice, an outbred strain, showing that its immunogenic backdrop are not exclusive for BALB/c mice haplotype.

An interesting consequence of our experiments was that not only the depletion of CD4⁺ T cells, but also of CD8⁺ T cells, securely afflicted the consecration of the anti-idiotypic response. The depletion of the CD4⁺ or CD8⁺ T cells earlier the first dose of P3 abrogated the antibody response, suggesting an important role for these cells at the kickoff stages of the anti-idiotypic response. Although historically CD8⁺ T cells have been regarded as cytotoxic cells, several studies take shown their chapters to actuate the immune responses, providing help for DC (Nakamura et al., 2007), NK (Shanker et al., 2007), and B cells (Cronin et al., 1995). Information technology has become articulate the existence of CD8⁺ T cell subsets analogous to the Th1 and Th2 CD4 counterparts in mice (Croft et al., 1994; Cronin et al., 1995; Sad et al., 1995) and humans (Salgame et al., 1991; Maggi et al., 1994; Birkhofer et al., 1996).

On the other mitt, the antibody response in BALB/c mice deficient of CD8⁺ T cells, immunized with P3 mAb emulsified in Freund's adjuvant, does not vary significantly with respect to the response in the mice with a normal CD8⁺ T prison cell population (information non shown). The activeness of the adjuvant to enhance the immune response, past activating dendritic cells (Salem et al., 2006; Agger et al., 2008), might annul the effect caused by the deficiency of CD8⁺ T cells. These results suggest that in the induction of an anti-P3 antibody response, CD8⁺ T cells could play a role equivalent to that of an adjuvant, probably as activators of the antigen presenting cells or straight activating the anti-idiotypic antibody secreting B cell (Lu et al., 2002). Another possible role of CD8⁺ T cells could be to eliminate or suppress some regulatory cell populations (Hahn et al., 2005), that could inhibit the induction of the antibody response against P3 mAb. Although the interaction between Id+ B prison cell and CD4⁺ T cells have been previously published (Weiss and Bogen, 1991; Bogen and Weiss, 1993; Munthe et al., 1999; Jacobsen et al., 2010), to our knowledge, this is the outset report that involves CD8⁺ T cells in the induction of an anti-idiotypic response in vivo. This result opens new possibilities for the understanding of the regulation and activation of B cell–T cell idiotypic networks.

Due to the importance of CD8⁺ T cells for the humoral response against P3, and knowing that P3 mAb is able to induce T cells proliferation in vitro (Perez et al., 2002), we studied if this antibiotic could take some affect on the CD8⁺ T cell population in vivo. Beginning nosotros depleted the CD8⁺ populations in mice that were treated with P3 mAb or an irrelevant control antibody. In fact, the percentage of CD8⁺ T cell recovery in CD8⁺ cells depleted mice previously immunized with P3 was significantly higher than the one detected in the animals treated with the control antibody. This result demonstrated that P3 mAb has the ability to aid the recovery of the CD8⁺ T population in vivo. This could be a consequence of a proliferative outcome over the remnant or new emerging CD8⁺ T cells or due to the consecration of a greater resistance to depletion past the cytotoxic antibody. Since the anti-CD8a antibody used cause complement dependent prison cell death (Cobbold et al., 1984), the consecration of CD8⁺ T cell proliferation due to immunization would be the near suitable explanation.

In order to evaluate the functionality of the expanded T cells and the chapters of P3 mAb to reactivate an immunosuppressed immune system, we used an in vivo model. In this model BALB/c mice go susceptible to an allogeneic tumor, which would be rejected in immunocompetent mice, due to the immunosuppression induced past the inoculation of a syngeneic tumor. In our experiment, 50% of the BALB/c mice inoculated with the syngeneic chest tumor F3II showed B16-F10 allogeneic tumor growth, in contrast with but 19% on P3 treated mice. This result confirms that P3 mAb is able to recover functional T cells in situations of immunosuppression (Sauer et al., 2008).

Next we evaluated the chapters of P3 mAb to induce the recovery of T cell populations in a clinically relevant scenario, like the immunosuppression induced by cytotoxic drugs during cancer treatments. Nosotros could ostend that in mice immunosuppressed due to cyclophosphamide handling, which affect both T and B cell populations (Zuluaga et al., 2006), the handling with a single dose of P3 mAb likewise induced a meaning increase in the recovery of CD4⁺ and CD8⁺ T. The same experiment performed in C57BL/6 mice did not evidence pregnant differences between the P3 mAb immunized and non-immunized mice (information not shown). This result confirms the ability of P3 mAb to induce T lymphocytes proliferation in BALB/c mice.

The results in this work ostend the high immunogenicity of P3 mAb and proved the participation of CD8⁺ T cells during the induction phase of the immune response. Moreover, we showed that the immunization with P3 mAb reconstitutes in vivo T cell populations and better immunosuppressed states in lymphopenic BALB/c mice. A fast reconstitution of T jail cell dependent amnesty is a critical effect for patients treated with lymphodepleting regimens, which tin can produce higher susceptibility to infections, reduced response to vaccines and easier tumor relapse (Nordoy et al., 2001; Parkman et al., 2006; Aoki et al., 2012).

Some molecules with immunorestorative properties are being evaluated, which include IL-7, IL-two, and IL-15. IL-seven has shown a stiff immunorestorative and vaccine adjuvant effects in preclinical studies. Several clinical trials with IL-7 are ongoing in settings of acquired immunodeficiency, chronic viral infection, and cancer, showing and then far to be well tolerated and to induce immunorestoration, preferential of naïve T cells (Mackall et al., 2011). IL-two has been used to boost effector immune responses in patients with cancers and infectious diseases and it is registered for the treatment of renal-cell carcinoma. Nonetheless, the response to the treatment is achieved in less than 10% of the patients, probably because this cytokine stimulates regulatory T cells, which could suppress antitumor responses (Lemoine et al., 2009).

IL-15 is a potent proinflammatory cytokine, with antitumor furnishings in several preclinical mouse tumor models. However, administration of IL-15 solitary was non optimal because it also activated allowed organisation negative regulatory checkpoints that might affect the immune response (Yu et al., 2010).

In this commodity nosotros show the starting time evidences that P3 mAb could ameliorate immune restoration, peculiarly for T cells. To our knowledge, this is the kickoff written report of an immunogenic antibiotic with this capacity. The negative impact of lymphopenia for cancer patients and the fact that in this moment there is not a product that has totally prove its effectiveness in the dispensary; make it worthy to further study this important property of P3 mAb. Furthermore, P3 has an especial characteristic that differentiates information technology from the remainder of the molecules evaluated equally immune-stimulators: P3 information technology is an antibody and information technology would be possible the active induction of P3-similar antibodies sharing this property by anti-idiotypic immunization. In fact, 1E10 mAb, P3 mAb anti-idiotype has been used in several clinical trials in lung, melanoma, and breast cancer patients, showing that is able to induce a specific "P3-like" anti-NeuGcGm3 response (Alfonso et al., 2002; Diaz et al., 2003; Hernandez et al., 2008).

Experiments to further report the phenotypes and functions of the induced T prison cell populations in allowed compromise mice as well as to farther empathise the role of CD8⁺ T cells in the activation of immune networks are ongoing.

Materials and Methods

Animals

Female person BALB/c, C57BL/6, and NMRI mice, 6–8 weeks old, were purchased from the Center for Laboratory Animal Production (CENPALAB, Havana, Cuba). Animals were housed and bred in a bulwark maintained room according to the guidelines stipulated by the Animate being Subject Committee Reviews Board at the Center of Molecular Immunology (CIM). Animal studies were performed with approval from CIM'due south Institutional Fauna Care and Utilise Committee.

mAbs

P3 mAb (IgM, yard), recognizes NeuGc-containing gangliosides and sulfated glycolipids (Vazquez et al., 1995). P3 mAb was purified from ascitic fluid by gel filtration chromatography using a Sephacryl S-300 high-resolution column (Pharmacia) equilibrated with PBS containing 0.5 G NaCl. E1 mAb (IgM, one thousand, anti-GM2), was used equally isotype control. Anti-CD8a and anti-CD4a antibodies (IgG2b, k) were purified from supernatant of YTS 169.4.2.1 and YTS 191.ane.1.2, respectively (Cobbold et al., 1984).

Cells

Murine breast cancer F3II cells and melanoma B16-F10 cells were grown in RPMI-1640 medium (Gibco), supplemented with 10% fetal calf serum (FCS), 10 U/mL penicillin, and 10 mg/mL streptomycin. Tumor cells were counted and the viability was greater than 90%, as assayed by trypan blue exclusion test.

Immunization protocols

Induction of antibody response confronting P3 mAb

To evaluate the relevance of the immunization route for P3 mAb immunogenicity, BALB/c mice were immunized with iv biweekly dose of fifty μg of P3 mAb in PBS subcutaneous (s.c.), intramuscular (i.thou.), or intraperitoneally (i.p.). To evaluate the importance of the dose number and the presence of adjuvant, BALB/c mice were immunized with 1 or four dose of 50 μg of P3 mAb, at 2 weeks intervals, subcutaneously, in the presence or absence of Freund'southward adjuvant. C57BL/6 and NMRI mice were immunized with four dose of 50 μg of purified P3 mAb in PBS, at 2 weeks intervals, subcutaneously. Serum samples were taken before the commencement and vii days later on each immunization.

Antibiotic response induction against P3 mAb in CD8⁺ T cells depleted mice

To determine the moment at which CD8⁺ and CD4⁺ T cells participate in the induction of the anti-P3 mAb response, three groups of BALB/c mice were immunized four times at two weeks intervals, subcutaneously, with 50 μg of P3 mAb and were inoculated with 1 mg of anti-CD8a or anti-CD4 antibody intraperitoneally one time before the first, the second or the tertiary dose of P3 mAb immunization, respectively. P3 mAb immunized mice without anti-CD8a or anti-CD4 antibody treatment were used as control. Serum samples were taken before the first and 7 days later the fourth immunization.

P3 mAb consecration of in vivo stimulation of CD8⁺ T cells

BALB/c and C57BL/vi mice were immunized subcutaneously with 50 μg of P3 or E1 mAbs 4 times at 2 weeks interval. Three days afterward last immunization mice were inoculated with 1 mg of anti-CD8a antibody intraperitoneally followed by two daily doses of 50 μg of P3 or E1 mAbs, intravenously. Finally, 24 h later mice were sacrificed and their inguinal lymph nodes were extracted to clarify the number of CD8⁺ T cell past menstruum cytometry.

Allogeneic tumor rejection protocol

BALB/c mice were immunized 4 times at 2 weeks intervals, subcutaneously, with 50 μg of P3 mAb. Viii days later the start P3 mAb immunization mice were inoculated with chest tumor prison cell line F3II (two × x⁵ cells per mouse, subcutaneously) and 21 days later with the allogeneic melanoma cell line B16-F10 (5 × ten⁵ cells per mouse, subcutaneously). C57BL/half-dozen and BALB/c mice inoculated just with melanoma B16-F10 cells were used as positive and negative control, respectively. The tumor size measurement started seven days after melanoma inoculation, twice a week. For ethical reasons, animals were sacrificed when the general condition of the animals was affected.

P3 mAb induction of lymphocytes population recovery later cyclophosphamide treatment

BALB/c mice were treated with two doses of cyclophosphamide (200 mg/kg) at 4 days interval. Two days later mice were inoculated with l μg of P3 or E1 mAbs, intravenously. Later 48 h mice were sacrificed and their inguinal lymph nodes were extracted, cell number was counted used Neubauer's chamber. Percentage of each lymphocytes population (CD4⁺ T, CD8⁺ T, and B cells) were measured by flow cytometry, taken PBS treated group equally 100%.

ELISA

Solid phase ELISA was performed as previously described (Reitan and Hannestad, 2002), using 96-well polystyrene MaxiSorp microtiter plates (Nunc) which were coated with 10 μg/mL of purified P3 mAb and incubated with preimmune or hyperimmune sera dilute 1:100. Element of group i phosphatase-conjugated caprine animal anti-mouse IgG (Fcγ-specific, Jackson Immunoresearch Laboratories) was used equally second antibody. The reaction was developed with p-nitro-phenyl phosphate substrate (Sigma) in diethanolamine buffer (pH ix.8). The absorbance was measured at 405 nm in an ELISA reader (Organon Teknika).

Period cytometry

Cells were obtained from inguinal lymph nodes and were incubated with PE-conjugated anti-mouse CD8a, FITC-conjugated anti-mouse CD4 and FITC-conjugated anti-mouse B220 (BD Pharmingen) for thirty min at 4°C and and then done with PBS twice. Cell surface antigen expression was evaluated by single- or double-immunofluorescence staining. Analysis was performed with a FACScan analyzer (Becton Dickinson, USA) and WinMDI 2.ix software.

Statistical analysis

Differences in IgG antibody response against P3 mAb were evaluated by Mann–Whitney examination. The comparison between the number of lymphocytes in the experimental groups treated in vivo with anti-CD8a antibody or cyclophosphamide were analyzed past Kruskal Wallis and Dunnett t-test. The differences in the percent of B16-F10 incidence was analyzed using Pearson's chi-foursquare (χ²) test. The differences were considered significant when p ≤ 0.05. All statistical tests were i-tailed, and conducted using SSPS for Windows version xi.v.1 software.

Disharmonize of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed equally a potential conflict of interest.

Acknowledgments

We give thanks to Yohan González from Experimental Immunotherapy Department for his work in animal care and manipulation.

Appendix

Figure A1

Kinetic of IgG antibodies response in BALB/c mice. Mice were immunized with one (1d) or iv (4d) doses of P3 mAb or an isotype command mAb in PBS or emulsified in Freund's adjuvant (FA). Humoral response were tested confronting P3 mAb (A,C) or isotype command mAb (B,D). The claret samples were obtained before the immunization and on days 7, 21, 35, and 49 of the immunization protocol. Sera were diluted 1:100 and the reactivity confronting purified P3 mAb was assessed by ELISA. Binding was adamant using alkaline phosphatase-conjugated caprine animal anti-mouse IgG (Fcγ-specific). Values are expressed as means ± SD (n = 5).

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