The restricted expression of granzyme M in human lymphocytes

We have analyzed the expression of human granzyme M (Gzm M) in various human leukocyte subsets using the specific mAb 4H10. Using FACS and Western blotting analysis we compared the expression of Gzm M with that of other granzymes (Gzm A and Gzm B) and the lytic protein perforin. Human Gzm M was constitutively highly expressed in NK cells as was perforin and Gzm A. Surprisingly, freshly isolated NK cells had very low (sometimes undetectable) levels of Gzm B. In contrast to Gzm B and perforin, Gzm M was not detected in highly purified CD4(+) and CD8(+) T cells either constitutively or after short term activation in vitro. However, low levels of Gzm M were observed in some T cell clones on prolonged passage in vitro. Gzm M was not detected in highly purified neutrophils, monocytes, or tumor cells of the myelomonocytic lineage. Examination of minor T cell subsets from human peripheral blood showed detectable Gzm M in CD3(+), CD56(+) T cells and gammadelta T cells. A histological staining procedure was developed that demonstrated a granular staining pattern for Gzm M and a cellular distribution similar to that observed by Western blotting. These data indicate that the expression of Gzm M does not always correlate with the lytic activity of cytotoxic cells. However, expression of Gzm M in NK cells, CD3(+), CD56(+) T cells, and gammadelta T cells suggests that this enzyme may play some role in innate immune responses.     

Perforin, granzyme B, and FasL expression by peripheral blood T lymphocytes in emphysema

Background

It is generally accepted that emphysematous lungs are characterized by an increase in the numbers of neutrophils, macrophages, and CD8⁺ T lymphocytes, the lasts having increased cytotoxic activity. Because systemic inflammation is also a component of emphysema, we hypothesize that peripheral CD8⁺ T lymphocytes of emphysematous smokers who show evidence of systemic inflammation will have higher expression of cytotoxic molecules.

Methods

We assessed parameters of systemic inflammation in normal individuals (smokers or non-smokers) and in emphysematous subjects with an active smoking history by measuring serum interleukine-6, C-reactive protein, and tumor necrosis factor. Expression of perforin, granzyme B, and FasL protein by CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and natural killer cells were assessed by flow cytometry while perforin, granzyme B, and FasL mRNA expression were measured on purified systemic CD8⁺ T lymphocytes by real-time PCR.

Results

Emphysematous smokers had higher levels of serum interleukine-6 than normal subjects. Even with the presence of systemic inflammation in emphysematous smokers, the percentage of peripheral CD8⁺ T lymphocytes, CD4⁺ T lymphocytes, and NK cells expressing perforin and granzyme B protein was not different between the three groups.

Conclusion

Despite evidence of systemic inflammation, peripheral T lymphocytes of emphysematous smokers did not show higher levels of cytotoxic markers, suggesting that increase of activated T lymphocytes in the emphysematous lung may be due to either activation in the lung or specific peripheral recruitment.     

Activated mouse B cells lack expression of granzyme B

Recently, it has been reported that human B cells express and secrete the cytotoxic protease granzyme B (GrB) after stimulation with IL-21 and BCR cross-linking. To date, there are few clues on the function of GrB in B cell biology. As experimental transgenic murine systems should provide insights into these issues, we assayed for GrB in C57BL/6 B cells using an extensive array of physiologically relevant stimuli but were unable to detect either GrB expression or its proteolytic activity, even when Ag-specific transgenic BCRs were engaged. Similar results were also obtained with B cells from DBA/2, CBA, or BALB/c mice. In vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of GrB in CTLs, but not in B cell populations. We also investigated a possible role of GrB on the humoral immune response to the model Ag 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin, but GrB-deficient mice produced normal amounts of Ab with typical affinity maturation and a heightened secondary response, demonstrating conclusively the redundancy of GrB for Ab responses. Our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans. The physiological consequences of GrB expression in human B cells remain unclear, and the current study suggests that experimental mouse models will not be helpful in addressing this issue.     

Differential regulation of voltage- and calcium-activated potassium channels in human B lymphocytes.

The expression and characteristics of K+ channels of human B lymphocytes were studied by using single and whole-cell patch-clamp recordings. They were gated by depolarization (voltage-gated potassium current, IKv, 11-20 pS) and by an increase in intracellular Ca2+ concentration (calcium-activated potassium current, IKCa, 26 pS), respectively. The level of expression of these channels was correlated with the activational status of the cell. Both conductances are blocked by tetraethylammonium, verapamil, and charybdotoxin, and are insensitive to apamin; 4-aminopyridine blocks IK, preferentially. We used a protein kinase C activator (PMA) or antibodies to membrane Ig (anti-mu) to activate resting splenocytes in culture. Although IKv was recorded in the majority of the resting lymphocytic population, less than 20% of the activated cells expressed this conductance. However, in this subset the magnitude of IKv was 20-fold larger than in resting cells. On the other hand, IKCa was detected in nearly one half of the resting cells, whereas all activated cells expressed this current. The magnitude of IKCa was, on average, 30 times larger in activated than in nonactivated cells. These results probably reflect that during the course of activation 1) the number of voltage-dependent K+ channels per cell decreases and increases in a small subset and 2) the number of Ca(2+)-dependent K+ channels per cell increases in all cells. We suggest that the expression of functional Ca(2+)- and voltage-activated K+ channels are under the control of different regulatory signals.     

Human B cells differentiate into granzyme B-secreting cytotoxic B lymphocytes upon incomplete T-cell help

Recently, CD4(+) T helper cells were shown to induce differentiation of human B cells into plasma cells by expressing interleukin (IL-)21 and CD40 ligand (CD40L). In the present study we show, that in the absence of CD40L, CD4(+) T cell-derived IL-21 induces differentiation of B cells into granzyme B (GzmB)-secreting cytotoxic cells. Using fluorescence-activated cell sorting (FACS) analysis, ELISpot and confocal microscopy, we demonstrate that CD4(+) T cells, activated via their T-cell receptor without co-stimulation, can produce IL-21, but do not express CD40L and rapidly induce GzmB in co-cultured B cells in an IL-21 receptor-dependent manner. Of note, we confirmed these results with recombinant reagents, highlighting that CD40L suppresses IL-21-induced GzmB induction in B cells in a dose-dependent manner. Surprisingly, although GzmB-secreting B cells did not express perforin, they were able to transfer active GzmB to tumor cell lines, thereby effectively inducing apoptosis. In contrast, no cytotoxic effects were found when effector B cells were activated with IL-2 instead of IL-21 or when target cells were cultured with IL-21 alone. Our findings suggest GzmB(+) cytotoxic B cells may have a role in early cellular immune responses including tumor immunosurveillance, before fully activated, antigen-specific cytotoxic T cells are on the spot. CD40 ligand determines whether IL-21 induces differentiation of B cells into plasma cells or into granzyme B-secreting cytotoxic cells.     

Serglycin-deficient cytotoxic T lymphocytes display defective secretory granule maturation and granzyme B storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.     

Design of human granzyme B variants resistant to serpin B9

Human granzyme B (hGB) is a serine protease involved in immune‐mediated apoptosis. Its cytotoxicity makes it potentially applicable in cancer therapy. However, the effectiveness of hGB can be hampered by the cytosolic expression of a natural protein inhibitor, human Serpin B9 (hSB9). Here, we used computational approaches to identify hGB mutations that can affect its binding to hSB9 without significantly decreasing its catalytic efficiency. Alanine‐scanning calculations allowed us to identify residues of hGB important for the interaction with hSB9. Some variants were selected, and molecular dynamic simulations on the mutated hGB in complex with hSB9 in aqueous solution were carried out to investigate the effect of these variants on the stability of the complex. The R28K, R201A, and R201K mutants significantly destabilized the interaction of the protein with hSB9. Consistently, all of these variants also retained their activity in the presence of the Serpin B9 inhibitor in subsequent in vitro assays of wild‐type and mutated hGB. In particular, the activity of R201K hGB with and without Serpin B9 is very similar to that of the wild‐type protein. Hence, R201K hGB emerges as a promising species for antitumoral therapy applications. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Expression, refolding, and purification of recombinant human granzyme B

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.     

Development and regulation of chlamydia-responsive murine B lymphocytes

We have examined characteristics of chlamydia-stimulated mouse B cells as well as cells that regulate polyclonal responses in vitro. B lymphocyte proliferation stimulated by chlamydia arises at a similar time as Escherichia coli lipopolysaccharide (LPS)-induced proliferative responses during ontogeny. In contrast, development of immunoglobulin (Ig)-secreting cells after chlamydia stimulation is delayed by several weeks relative to ontogeny of LPS-inducible plaque-forming cells (PFC). The lack of Ig secretion by immature B cells is not due to a deficiency of Lyb5+ B lymphocytes, since X-linked immunodeficient (xid) NBF1 mice that lack this B lymphocyte population respond well to chlamydia stimulation. Adherent cells are important for chlamydia-stimulated B lymphocyte differentiation, but are not as necessary for their proliferation. Neither adult adherent cells nor T cells can correct the inability of immature spleen cells to develop into Ig-secreting cells; spleen cells from 2-wk-old mice (i.e., immature B cells) will not suppress adult B lymphocyte responses to chlamydia. When B lymphocytes are separated according to their buoyant densities, chlamydia stimulates low density (activated) B cells to proliferate and differentiate better than high density (resting) cells. Proliferative responses to chlamydia arise earlier during ontogeny, do not require adherent cells, and can proceed to a relatively greater extent in resting B cell population (compared with activated B cells) than induction of Ig-secreting cells.     

LFA-1 membrane molecule in the regulation of homotypic adhesions of human B lymphocytes

We report here that MAb to human LFA-1 inhibit spontaneous homotypic adhesions of human B lymphocytes. This is, to our knowledge, the first report of a MAb that inhibits human homotypic intercellular adhesions for any cell type. LFA-1 has previously been recognized as a molecule capable of regulating specific immunologic adhesions between T lymphocytes and antigen-bearing target cells. The present findings show that the role of LFA-1 is not limited to adhesions initiated by specific immunologic recognition. The results indicate that the LFA-1 molecule is capable of regulating lymphocyte adhesions, possibly because it is a direct participant in adhesion formation.     

Discordant expression of human Ia-like antigens on hematopoietic progenitor cells

The expression of HLA-DR, SB, MT2, and DC antigens on human hematopoietic progenitor cells has been determined by using monoclonal antibodies with complement (C)-mediated cell lysis and immune separation techniques. HLA-DR was detected on greater than 85% of CFU-G/M, myeloid clones (MyCl), BFU-E, and CFU-E. CFU-E were less susceptible to C-mediated lysis at suboptimal C concentrations. The polymorphic MT2 and SB antigens were also present on all categories of progenitor cells, although a lesser proportion of cells were positive. Because in most individuals the antigen density of MT2 and SB, as determined by monoclonal antibody staining, was also lower on B cells and monocytes when compared to HLA-DR expression, the lower number of positive progenitor cells probably reflects lower antigen density rather than distinct positive and negative progenitor cell populations. The DC antigen is expressed weakly on monocytes and B cells, although there is considerable individual variation. In some individuals, distinct DC-positive and -negative monocyte populations are detectable. The DC antigen was not detected on myeloid progenitor cells, even in those individuals with moderate DC expression on their monocytes and B cells. This discordant expression of DC and other Ia-like antigens on hematopoietic progenitor cells may be of physiologic significance and may assist in the purification of progenitor cells from blood and marrow.     

Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C-null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8(+) cytotoxic T lymphocyte (CTL) raised in cathepsin C-null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C-null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.     

Structural organization of the hCTLA-1 gene encoding human granzyme B

Cytotoxic T lymphocytes (CTLs) and natural killer/lymphokine-activated cells produce granzymes, a family of serine esterase proteins located in cytoplasmic granules. These might be involved in different cytotoxic pathways. We report the structural organization of the human gene encoding granzyme B (hCTLA-1). A 4.75-kb genomic DNA fragment containing all the sequences of granzyme B-encoding cDNA clones has been sequenced. The gene is composed of five exons and four introns. A comparison with the genomic organization of murine CCP1/CTLA-1 showed very similar structure and a 76% nucleotide homology in the coding sequences. This suggests that both genes may have a common ancestor. No typical regulatory element was detected in the 1160 bp upstream from the ATG start codon. The detection of a second locus related to hCTLA-1 is also described.     

Antigen-dependent regulation of interleukin 2 receptor expression on cloned human cytotoxic T lymphocytes

IL 2 receptor expression as a function of time after antigenic stimulation was examined on antigen-dependent human CTL clones specific for type A influenza virus. The anti-Tac monoclonal antibody was used to follow IL 2 receptor levels on the cloned cells. Shortly after antigenic stimulation, IL 2 receptor expression was maximal; by 1 wk, however, levels had decayed considerably, and by 2 wk only background expression remained. Reexpression of IL 2 receptors could be induced by exposure of quiescent clones to antigen or lectin. IL 2-driven proliferation of the cytotoxic clones was also examined, and it decayed with the same kinetics as IL 2 receptor levels. Proliferation of quiescent cells could also be obtained by antigen-specific stimulation. Thus, IL 2 receptor expression by human CTL clones at least in part regulates the antigen-specific proliferation of these cells.     

Differential regulation of interleukin-1 gene expression in human CD3- large granular lymphocytes

Our laboratory analyzed the expression of lymphokine and cytokine mRNA in CD3- peripheral blood large granular lymphocytes (LGL). Herein we present evidence that this subset of lymphocytes can synthesize IL-1 beta mRNA constitutively and that the cytoplasmic mRNA levels of IL-1 beta can be increased rapidly by interleukin (IL)-2. IL-1 alpha mRNA is expressed constitutively very infrequently and increases in IL-1 alpha mRNA are seen only after prolonged incubation with IL-2. Furthermore, IL-1 activity could not be detected in LGL culture supernatants, indicating that other processes may be involved in releasing biologically active IL-1 from LGL. In addition, MAb to the p75 IL-2 receptor on LGL abrogated IL-2 induction of IL-1 beta mRNA, suggesting that IL-2 signaling via the p75 IL-2 receptor induced IL-1 beta gene expression in LGL. Since, in contrast to T cells, LGL are capable of mediating effector functions without prior stimulation, they are said to be already "primed" for response. Overall, these data suggest that constitutive lymphokine gene expression may be involved in the in vivo priming of LGL.     

Heterogeneity and function of human B lymphocytes

B lymphocytes represent an important arm of the immune system. Besides their main function of providing antibodies protecting against pathogens, they also exert some regulatory functions, in particular for secondary lymphoid tissue differentiation. Human B cells can be divided in various subsets representing different maturation stages and different pathways of humoral immune responses. Na?ve IgMlow IgDhigh CD27- B cells can participate in T-cell dependent immune responses leading to germinal center formation in follicles of secondary lymphoid organs. Interactions with follicular helper T cells, a recently identified CD185+ T cell population providing help to follicular B cell, involve costimulatory molecules including CD40, CD27, CD278 and SAP-recruiting receptors. B cell interaction with follicular helper T cells represents a critical step controlling the generation of plama cells that ultimately produce high affinity, somatically mutated, class-switched antibodies or of their memory B cell counterpart (identified as CD27+ Ig switched or IgMonly B cells). IgMhigh IgDlow CD27+ B cells are a puzzling population apparently specialized in T-independent responses to bacterial capsular polysaccharides. The extra-follicular, probably antigen-independent, differentiation pathway of these cells, allowing pre-immune repertoire diversification by somatic hypermutation, is not yet characterized. However, circulating IgMhigh IgDlow CD27+ B cells are similar to splenic marginal zone B cells. In addition to these subsets, minor populations can also be identified in peripheral blood, such as transitional B cells and plasma blasts. All together, deciphering human B cell heterogeneity provides tools for investigations of humoral immunodeficiencies and auto-immune diseases, that will in return shed more light on B cell biology.