Blood, 1 May 2002, Vol. 99, No. 9, pp. 3479-3481
CORRESPONDENCE
To the editor:
Sperm protein 17 is not expressed on normal leukocytes
Sperm protein 17 (Sp17) is thought to promote heparan
sulfate-mediated cell-cell adhesion.1,2 Recent works
indicated the presence of Sp17 transcripts in tumor
cells.2-4 Since Sp17 mRNA is detected only within testis,
Sp17 could be an ideal target for tumor vaccine. This notion is
supported by our ability to generate Sp17-specific cytotoxic
T-lymphocytes (CTLs) capable of lysing HLA-matched fresh tumor targets
from peripheral blood mononuclear cells of healthy and
cancer-bearing patients.5,6 A recent study using a Sp17
polyclonal rabbit antisera in flow cytometry suggested that Sp17 might
be a ubiquitous protein expressed on both normal and malignant
leukocytes.2 Further clarification into the normal tissue
distribution of Sp17 protein is therefore of vital importance. To
address this, we have used a recombinant Sp17 protein we
produced5 to immunize a BALB/c mouse to produce Sp17 mouse monoclonal antibodies (MoAbs) from the immunized
splenocytes. Successful generation of the Sp17 mouse MoAbs was first
confirmed in Western blot analysis by demonstration of the ability of
the antibody to bind the recombinant Sp17 protein and not a control recombinant protein produced using an identical route (Figure 1A). The antibodies are mouse IgG1
in subclass.
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| Figure 1.
(A) Western blot analysis confirming the successful generation of Sp17
mouse monoclonal antibodies, showing binding of the antibodies to a
recombinant Sp17 protein and not to a control recombinant protein. Both
recombinant proteins were Escherichia coli-derived and
contained a C-terminal 6-His tag. (i) Coomassie blue staining of a 10%
sodium dodecyl sulfate-polyacrylamide gel. (ii) Western blot
analysis demonstrating the binding of Sp17 mouse monoclonal antibodies
to only recombinant Sp17 protein (M = molecular weight marker; lane
1 = recombinant Sp17 protein; lane 2 = control recombinant
protein). (B) Sp17 protein was detected on normal spermatozoa (i and
iii) but not on any of the peripheral blood leukocytes (ii and
iv) by either immunofluorescence cytology (i and ii) or
immunocytochemistry (iii and iv). (C) Immunohistochemistry showing the
expression of Sp17 protein in normal testis (i). In contrast, Sp17
protein was not detected in either normal tonsils (ii) or Peyer
patches (iii).
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The antibodies were then applied in immunocytochemistry to
determine Sp17 protein expression in normal peripheral blood cells. We
found that, unlike in normal spermatozoa, Sp17 was not detected either
on the cell surface or in the cytoplasm of normal peripheral blood
lymphocytes, monocytes, or granulocytes (Figure 1B). This finding was
further confirmed using immunofluorescence microscopy (Figure 1B) that
showed the lack of Sp17 protein expression on normal peripheral blood
leukocytes. These results therefore do not support the recent findings
by Lacy and Sanderson,2 who used Sp17 rabbit polyclonal
antisera in flow cytometry that suggested Sp17 might be a
protein ubiquitously expressed in normal leukocytes. The expression of
Sp17 within human Peyer patches was next investigated. A study
showed the up-regulation of bovine Sp17 mRNA within the Peyer patches
following bovine rotavirus infection of fetal lamb.7 This
study raised the possibility that Sp17 may be expressed in small intestine, an anatomic site that was not included in the panel of
normal tissue we previously studied.3 Interestingly, in
contrast to the mRNA findings in fetal lamb, Sp17 protein was not
detected within the human intestinal Peyer patches (Figure 1C). In
addition, Sp17 protein also was not detected in any cells, including
plasma cells, in the normal tonsils (Figure 1C). A control staining for Sp17 using normal testis showed strong expression of Sp17
(Figure 1C). Our results therefore indicate that Sp17 is not expressed
on normal leukocytes.
There are many reasons that may account for the discrepancy
between our results and those reported by Lacy and Sanderson
on the expression of human Sp17 on normal leukocytes. It is
well-recognized that rabbit polyclonal antisera are not always suitable
for use in flow cytometry because of the difficulty in the selection of an appropriate control serum. Although preimmuned rabbit serum was used
as a negative control by Lacy and Sanderson, the proportions of the different immunoglobulin isotypes within the reagents, and hence
the "nonspecific stickiness" of the reagents, may not be identical.
This, together with the extreme sensitivity of flow cytometry, may have
resulted in positive signals observed in normal peripheral leukocytes
reported by Lacy and Sanderson. Ideally, their results should
have been confirmed by immunocytochemistry and not reverse
transcriptase-polymerase chain reaction (RT-PCR), because
RT-PCR is more suited as a rapid mRNA screening method rather than as a
method for confirming protein expression.
Based on tumor specificity, Sp17 remains an ideal target for
cancer immunotherapy. This is supported by our previous results indicating the restricted normal tissue expression of human Sp17 mRNA
and the findings in the present study showing that Sp17 protein is not
expressed in normal human leukocytes. The apparent lack of adverse
reactions in healthy males who develop immunity against Sp17
following vasectomy further provides the data supporting the in
vivo safety of targeting Sp17 in tumor vaccines. Our findings of the
lack of expression of Sp17 in normal leukocytes are not surprising,
since we previously found that Sp17-primed CTLs did not kill
HLA-matched Epstein Barr-virus-transformed lymphoblastoid cells and peripheral blood monocyte-derived dendritic cells.
Fabio Grizzi, Seah H. Lim, Maurizio Chiriva-Internati, Barbara Franceschini, Zhiqing Wang, David Lawrence, and Nicola Dioguardi
Correspondence: Seah H. Lim, Don and Sybil Harrington
Cancer Center, 1500 Wallace Blvd, Amarillo, TX 79106; e-mail:
slim{at}harringtoncc.org
Acknowledgments
Supported by the National Institute of Health/National Cancer
Institute (RO1 CA 88434), the Cancer Treatment Research Foundation, and
the Mary Kay Ash Charitable Foundation.
References
1.
Wen Y, Richardson RT, Widgren EE, O'Rand MG.
Characterization of Sp17: a ubiquitous three-domain protein that binds heparin.
Biochem J.
2001;357:25-31[CrossRef][Medline]
[Order article via Infotrieve].
2.
Lac HM, Sanderson RD.
Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfate-mediated cell-cell adhesion.
Blood.
2001;98:2160-2165[Abstract/Free Full Text].
3.
Dong G, Loukinova E, Smith CW, Chen Z, van Waes C.
Genes differentially expressed with malignant transformation and metastatic tumor progression of murine squamous cell carcinoma.
J Cell Biochem.
1997;29(suppl):90-100[CrossRef].
4.
Lim SH, Wang Z, Chiriva-Internati M, Xue Y.
Sperm protein 17 is a novel cancer-testis antigen in multiple myeloma.
Blood.
2001;97:1508-1510[Abstract/Free Full Text].
5.
Chiriva-Internati M, Wang Z, Xue Y, Bumm K, Hahn AB, Lim SH.
Sperm protein 17 (Sp17) in multiple myeloma: opportunity for myeloma-specific donor T cell infusion to enhance graft-versus-myeloma effect without increasing graft-versus-host disease risk.
Eur J Immunol.
2001;31:2277-2283[CrossRef][Medline]
[Order article via Infotrieve].
6. Chiriva-Internati M, Wang Z, Salati E, Timmins P, Lim SH. Tumor vaccine
of ovarian cancer targeting Sperm protein 17 (Sp17). Cancer. In
press.
7.
Tatlow D, Brownlie R, Babiuk LA, Griebel P.
Differential display analysis of gene expression during the induction of mucosal immunity.
Immunogenetics.
2000;52:73-80[Medline]
[Order article via Infotrieve].
Response:
Expression of Sp17 on normal tissues
We agree with Grizzi et al that clarification into the normal
tissue distribution of Sp17 protein is of vital importance, especially
because of their interest in using Sp17 as a target for immunotherapy.
We also agree that flow cytometry is extremely sensitive. We too were
concerned about false-positive staining, and that is precisely why, in
our published study,1 the appropriate controls were
included and expression confirmed by reverse transcriptase-polymerase chain reaction of Sp17 on normal leukocytes. We found that
levels of expression of Sp17 on normal leukocytes were relatively low but very similar to the low levels of expression seen on myeloma cells
by flow cytometry as reported by both us and the authors of the above
letter using the same rabbit antisera.1,2
In light of the comments by Grizzi et al, it is important to remember
that the absence of detection of an antigen by immunofluorescence cytology or immunocytochemistry does not prove that the antigen is not
present, only that it is not detected. Their inability to detect Sp17
on normal leukocytes could result from their monoclonal being of low
affinity, or from masking of the antibody epitope at the cell
surface. These possibilities are underscored by the relatively weak
staining of sperm that they show in Figure 1B (panel i).
But in reference to using Sp17 as a target antigen in
immunotherapy, the broader and more critical question concerns the
expression of Sp17 on somatic cells. Important work regarding this
issue was recently published by the laboratory of Dr Michael O'Rand, the discoverer of Sp17 and the leading authority on this
molecule.3 They conclude, "Although Sp17 expression is
highest in the testis, it is present in all of the mouse somatic
tissues examined and is highly conserved throughout all mammalian
species." Although Grizzi et al are obviously aware of this work,
they appear to ignore the conclusion by O'Rand's group that Sp17 is
ubiquitously expressed.
Lastly, the intent of immunotherapy using Sp17 as a target
antigen is to generate a massive cytotoxic T-lymphocyte
response against cells that express Sp17. This intense immune response may have pathological consequences distinct from the apparent benign
response of vasectomized men to circulating antibodies against Sp17.
Thus, to use the finding that there is a lack of adverse reactions in
healthy males who develop circulating antibodies against Sp17
as evidence supporting the in vivo safety of targeting Sp17 for
immunotherapy is dubious at best.
Ralph D. Sanderson and H. Marie Lacy
Correspondence: Ralph D. Sanderson, Arkansas Cancer Research
Center, University of Arkansas for Medical Sciences, Dept of
Pathology-Slot 517, Little Rock, AR
References
1.
Lacy HM, Sanderson RD.
Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfate-mediated cell-cell adhesion.
Blood.
2001;98:2160-2165.
2.
Lim SH, Wang Z, Chiriva-Internati M, Xue Y.
Sperm protein 17 is a novel cancer-testis antigen in multiple myeloma.
Blood.
2001;97:1508-1510.
3.
Wen Y, Richardson RT, Widgren EE, O'Rand MG.
Characterization of Sp17: a ubiquitous three-domain protein that binds heparin.
Biochem J.
2001;357:25-31
