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In his own words: Leonard Herzenberg, The Immunologist Who Revolutionized Research, Dies at 81

Reporter: Aviva Lev-Ari, PhD, RN

SOURCE

http://www.herzenberglab.org/images/stories/herzenberglab/Publications/Reprints/LAH500.pdf

Annu. Rev. Immunol. 2004. 22:1–31

doi: 10.1146/annurev.immunol.22.012703.104727

Copyright c° 2004 by Annual Reviews. All rights reserved

First published online as a Review in Advance on December 16, 2003

GENETICS, FACS, IMMUNOLOGY, AND REDOX:

A Tale of Two Lives Intertwined

Leonard A. Herzenberg and Leonore A. Herzenberg

Genetics Department, Stanford University Medical School, Beckman Center, Stanford,

California 94305–5318; email: LenHerz@darwin.stanford.edu,

LeeHerz@darwin.stanford.edu

Key Words glutathione, B cells, flow cytometry, allotype, HIV

INTRODUCTION

We (Len and Lee Herzenberg) have worked separately and together for more than

50 years. This blending of independence and mutual reliance is reflected here as we

shift back and forth in telling the story of the laboratory we have led and the life we

have lived. The space provided for this chapter is very generous.Yet, calculated out,

it amounts to roughly 100 words per year for each of us. To make the most of this,

we have written an autobiography rather than a history. In many instances, we have

referred only briefly, or not at all, towork that had major influences on our thinking.

In addition, we have adopted a policy of naming the many students, fellows, and

collaborators with whom we have worked only by referring to our joint work with

them.We hope the reader realizes there would be no biography worth writing were

it not for the contributions made by these and all of our other colleagues.

THE CALTECH YEARS

LEN: There were nine professors in genetics in the California Institute of Technology

(Caltech) Biology Division when I arrived in 1952 as an entering graduate

student. They worked with different organisms and taught different areas of biology,

but they were united by a common theme—how genes are expressed and how

they influence the appearance, physiology, function, and behavior of the organism.

The Biology Division at that time was small—one three-story building housed

the entire faculty, about a dozen postdocs, an equal number of graduate students,

and a couple of undergraduate biology majors. Among the roughly 20 faculty

members (visiting or permanent), there were seven Nobelists in the making: George

Beadle, Max Delbruck, Ed Lewis, Renato Dulbecco, Roger Sperry, JamesWatson,

and Barbara McClintock. Linus Pauling, Caltech Chemistry chair and winner of

0732-0582/04/0423-0001$14.00 1

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two Nobel prizes, was in the next building, connected to ours by a much-used

corridor. Creative thinking and challenging discussion were the rule; research

productivity was the outcome.

The development of tools and techniques that removed barriers to experimentation

also played a central role in the Caltech Biology Division culture.We routinely

used the pH meter and DU spectrophotometer recently invented by former Caltech

chemistry professor Arnold Beckman. My thesis advisor, H. K. Mitchell, was an

extraordinary glass blower and tinkerer who worked with me on an electrophoresis

device. Most things couldn’t be bought, so we had to build them. As I think about

it now, the automated fly counter that Ed Lewis built probably sowed the seeds for

the Fluorescence-Activated Cell Sorter (FACS) that I developed some years later.

In the same way, looking back at the cross-discipline culture in the Caltech

Biology Division, I see the origins of the eclectic research goals that Lee and I

have pursued. Over the years, we have ranged broadly and drawn our students

and fellows into immunology studies as diverse as showing that H-2 antigens are

surface proteins, using immunoglobulin (Ig) allotypes and classical genetics to

define the Ig heavy chain (IgH) chromosome region, demonstrating IgH allelic

and haplotype exclusion in B cells, defining functional subsets of T and B human

and murine lymphocytes, cloning and sequencing lymphocyte surface markers,

identifying fetal cells in maternal circulation, understanding redox influences on

transcription factor activation, and doing clinical studies to characterize and treat

the glutathione deficiency in HIV infection. However, the twin themes of genetics

and somatic cell function that guided (and still guide) this work, and the love of

reading and talking about these diverse areas with people of different scientific

interests, are well rooted in Caltech tradition.

Political activism was also important at Caltech. Joseph McCarthy, the Senator

from Wisconsin who made a career of finding communists under every bed, was

threatening to disrupt academic and personal freedom. In response, we joined

Linus Pauling, Matt Messelson, George Streisinger, and Arthur Galston, as well

as other faculty, students, and fellows in establishing a Federation of American

Scientists chapter and in protesting this “witch hunt.” A portion of Lee’s and my

life ever since has been devoted to helping the United States be the kind of country

we want our children and children’s children to grow up in.

LEE: When Len left Brooklyn for Caltech, he was 21 and I was 17. Logically,

because he had three years of graduate work ahead of him and I had three years

to finish my undergraduate degree, we decided that we would marry when we

both finished school. However, logic couldn’t overrule the three-thousand-mile

distance, the loneliness, or the $3/minute (1953 dollars) the telephone company

charged young lovers just to say hello. By the end of the fall of 1952, Len urged

me to apply to schools near Caltech, and when I was admitted to Pomona College

in Claremont we set a wedding date for the coming summer.

Needless to say, our parents thought we were too young, too innocent, too poor,

and too crazy. They were probably right. But we got married anyway, with their

TWO LIVES INTERTWINED 3

blessings, and set off on an adventure that is as exciting today as it was the day we

piled our stuff into the car Len’s parents gave us and started the long drive across

the country to Pasadena.

At Caltech, I was as enchanted as Len was by how immersed people were in

their work. I had already selected biology as my major, but I had no idea how

interesting it could be. For the rest of the summer, until the fall term started at

Pomona, I went to the lab every day with Len. I attended seminars; I read in the

library; I learned how to help Len with experiments; sometimes I even had the

courage to ask questions of someone other than Len. It was just plain fun, and I

couldn’t get enough of it.

Pomona College, on the other hand, turned out to be a disappointment. It was

an excellent school. There is no question about that. The liberal arts courses were

wonderful. But the Biology Department was still teaching gram stains (to identify

bacteria) and grilling us on the anatomy of flowers and reptiles. Meanwhile, Jim

Watson had just brought the double helix back to Caltech and was teaching about

it while I was sitting in a classroom learning things useful only to a stodgy highschool

biology teacher.

I would have enrolled as a Caltech undergraduate, but women weren’t even

admitted to Caltech graduate programs (there were only a few women postdocs

and research associates). Nevertheless, the biology faculty believed that women

were educable and worth educating. By the beginning of the second semester of

the academic year, they worked out an auditing program for me in which I would

be treated, and graded, like a Caltech biology major. They allowed me to take

whatever courses I wanted to, and even found a part-time job for me so that Len

and I could afford to eat. For each course I completed, the professor gave me a letter

certifying that I had met the course requirements and received a grade (always As,

as it turned out). So, although I didn’t get formal credit, I managed to take classes

such as virology from Max Delbruck, bacteriology from Renato Dulbecco, and

immunology from Ray Owen. I learned how to think about science from these

teachers.

Perhaps the most formative event for me during our time at Caltech, though,

was a dinner with Barbara McClintock, who had recently come to the Biology

Department as a revered visiting professor. Len and I had decided to drive into Los

Angeles for a Chinese meal and were about to leave when we noticed that the only

light left on in the building was coming from Dr. McClintock’s door.We peeked in

and sawthat shewasworking alone. Then we drewback into the shadows, debating

whether we should tiptoe away and not interrupt her, or whether she might actually

like to take a break and join us. Finally, we screwed up our courage and asked her

if she would like to go. “I’d be delighted,” she replied, and off we went.

At dinner, I naively asked Dr. McClintock howshe made suchwonderful discoveries.

Her answer, simple and straightforward, became my long-standing rudder.

She said that in the course of her work, she occasionally got a surprising result

that could not be reconciled with existing theory. First, she would decide whether

she believed the “exception”—in other words, she could not see any technical or

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interpretive flaws that undermined it. Next, if she believed it, she would commit

it to memory and compare it with any other exceptions she had come across.

Ultimately, a constellation of exceptions would coalesce to yield a testable hypothesis

that, if validated by additional experimentation, would provide the basis

for extending or altering the current paradigm.

Len and I have never forgotten that dinner with Dr. McClintock. Somehow, it

must have been meaningful for her as well. When I next met her, some ten years

later, I introduced myself by name and started to say, “Dr. McClintock, you may

not remember me: : :” when she cut me off with, “Oh, you’re the people who took

me to that Chinese restaurant in L.A.,” and she greeted me thus ever after.

THE PARIS YEARS

Len defended his thesis in August 1955. We left immediately for Paris, where

Len had organized a postdoctoral fellowship with Jacques Monod at the Pasteur

Institute. My childhood friend who had recently returned to her native France met

us at the boat, found us a room in a student hotel in the Parisian Latin Quarter, and

introduced us to the life poor students lived in Paris. It was great! And so was the

laboratory at Pasteur.

LEN: Jacques Monod’s laboratory at Pasteur was physically separate but intellectually

allied with Andre Lwoff’s laboratory two floors above. Francois Jacob, the

third member of the trio later awarded the Nobel Prize for their seminal molecular

biology studies, worked in the Lwoff laboratory. The two laboratories lunched

together virtually every day at a single long table in a small atrium on an inner

Institute courtyard, where huge glass vessels rumored to have been used by Louis

Pasteur were stored.

Lunch was marvelous. It wasn’t a formal seminar, but conversation revolved

around science in the lab and the world at large. Findings were analyzed, theories

debated, visitors questioned. Every day was an intellectual feast.

There were also many first-person history lessons about the days beforeWorld

War II and what the war was like in France. Monod was a major figure in the

resistance against the Nazis. Francois Jacob had been to Algeria and North Africa

and participated with the Free French in the liberation of France. Georges Cohen,

one of the Monod senior scientists and still a close friend today, survived the war as

a Jew in France and talked about things he did in the Resistance.We heard stories

about how the laboratory hid Jewish scientists when the SS came knocking. It all

sounded very romantic in 1955, ten years after the war ended. But as Georges and

Jacques Monod often reminded us, it wasn’t much fun when it was happening.

I should mention that, like the Caltech Biology Division, the group at Pasteur

accepted Lee as an unofficial student. At the beginning, she was pregnant with our

first child and spent most of her time working with me. Later, she brought the baby

to the lab most afternoons and continued working.

TWO LIVES INTERTWINED 5

The Cradle ofMolecular Biology

This was a very exciting time at Pasteur. The characteristics of the ¯-galactosidase

(LacZ) operon were unfolding before our eyes as each new piece of work was

completed. Under Monod’s leadership, I contributed two pieces to the puzzle: I

showed that the galactoside-concentrating mechanism encoded by the permease

gene in the LacZ operon increases the internal concentration of inducers that

upregulate expression of the LacZ operon genes and thus is responsible for the

autocatalytic increase in LacZ induction. In addition, I showed that the inducers

are acetylated, rather than phosphorylated (as people had thought), during LacZ

induction, thus opening the way to adding (after I left Pasteur) the ¯-galactoside

acetylase gene to the LacZ operon. These and other findings led Monod, Jacob,

and Lwoff to the discovery of the LacZ operon, which laid the groundwork for

much of modern molecular biology. For this, they received the Nobel Prize.

While at Pasteur, I met Melvin Cohen, who later became a Salk Institute immunologist.

Mel had worked with Monod and returned to visit several times.

Conversations with him then, as always, were highly stimulating. His presence in

the Stanford Biochemistry Department was a key motivation in my decision to

move to Stanford when the opportunity arose several years after I left Pasteur.

LEE: Aside from birthing a baby and learning to balance being a mom with being

a scientist (albeit only a budding one), I don’t have too much to showfor my time at

Pasteur. I did do one independent piece ofwork, but itwasn’t well received. Monod

had several times said that the thiogalactosides that we used to induce expression

of LacZ operon genes were unnatural compounds that could not be digested by

bacteria. This didn’t seem right to “wise guy” me. So I went out and scooped

up some fresh Parisian soil, put it into a flask with minimal medium containing

thiogalactosides as the only carbon source, and put the flask into the cabinet under

the bench.

About a week later, the medium in the flask was cloudy, and a clear sulfur

smell wafted out when I opened the top. Something was clearly growing and

“knew how” to break down thiogalactosides. Excitedly waving the flask, I went to

Jacques’ office to show him my prize. He was quite surprised and, in a manner I

hope I have learned, graciously said he was pleased to be wrong in this case. Some

time later, however, everyone in the laboratory was ready to kill me. While we

never had to sterilize thiogalactoside stock solutions before my little experiment,

after I opened Pandora’s flask, all of the thiogalactoside stocks got contaminated.

From then on, they all had to be sterilized immediately after they were made!

This incident aside, I mainly spent my time at Pasteur helping Len. Because

I was rather sedentary during the first year, and because Len loved hands-on experimentation,

I took over much of the data recording, computation, and display

(plotting) that was needed. The work was tedious (slide rules were the closest thing

to computers at the time). However, it gave me the opportunity to do a preliminary

analysis of the data and try novel approaches to analyzing LacZ induction

kinetics. Len left this to me. He was more interested in developing methods and

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experiment designs that would enable clear conclusions without a lot of mathematical

interference. This division of labor, which reflects Len’s innate preference

for concreteness and my innate love for theory, remains with us even today.

THE NIH YEARS

Just about the time that Len and I were considering what to do after Pasteur, an

ominous postcard caught up with us. It had spent rather a long time traveling to

France by surface mail, and it announced that Len should have reported for active

duty in the U.S. Army several days before the postcard was loaded onto the slowest

boat on the Atlantic. Late or not, the postcard made it quite clear that Len had been

drafted!

We immediately went to Jacques Monod for advice. He was as adamant as

we were that it would be a pity to interrupt Len’s scientific career to serve in

a peacetime army. “Why don’t you consider going to the National Institutes of

Health, my boy? I have just had an inquiry from Harry Eagle looking for a fellow

for his laboratory.He should be able to arrange for you to serve in the Public Health

Service instead.”

LEN: This was a shock, but I was not displeased with the idea of going to Harry

Eagle’s laboratory. I had already been thinking about doing genetic studies with

mammalian somatic cells. What better place to learn how to grow cells than the

laboratory that had just developed Eagle’s medium? Leaving Pasteur and the Escherichia

coliworldwould not be easy. But the challenges presented by mammalian

studies would also be exciting. So, without further ado, I decided that I was lucky

to have the opportunity to carry a pipette rather than a gun for my country and

asked Jacques to write to Harry Eagle on my behalf.

It took several months to untangle the draft board and Public Health Service

mess and to wrap up my work in Paris. But by the summer of 1957, Lee, Berri (our

toddler), and I were settled in the Bethesda area, and I began work at the NIH.

Eagle’s laboratory operated with more of a top-down structure than Pasteur and

lacked some of the intellectual and scientific excitement I was used to. However,

my colleagues in the laboratory, notably Robert DeMars (now at the University of

Wisconsin) and James Darnell (now at Rockefeller University), were great.

“Captain Harry,” as we sometimes called Harry Eagle (to his face as well as

behind his back), was focused on determining the nutritional conditions necessary

to establish and maintain long-term cell lines. I was, too, because to do mutation

and selection studies I needed to establish conditions that would allow individual

clones to grow. My finding that adding pyruvate to Eagle’s medium was sufficient

to support clonal growth let me begin exploring drug resistance markers for genetic

studies. In addition, it led to the addition of pyruvate as a normal constituent of

the medium.

The Federation of American Scientists came back into our lives shortly after

we arrived in Washington, because the national office was only a short distance

TWO LIVES INTERTWINED 7

from the NIH. Picking up where we left off at Caltech, we volunteered to work on

the newsletter and do some administrative work for the organization. Ultimately,

we helped to reorganize the office and put the administrative oversight into the

hands of a liberal Washington, DC, law firm.

Lee and the Salmonella Histidine Operon

LEE: Afewweeks after we arrived at the NIH, Len ran into Bruce Ames, a Caltech

buddy who had just been appointed to a permanent staff position at the NIH. As

luck would have it, Bruce was looking for his first technician and I was looking

for my first job. Len made the match.

Poor Bruce. No one should have had to put up with me as a technician. I was

always asking “why” and looking for better ways to do things. As a graduate

student, I would probably have been fine. But as a technician responsible for doing

work that someone else gave me to do, and generating data that someone else was

supposed to interpret, I was clearly a pain in the neck. Nevertheless, Bruce put up

with me, and I learned to get the work done. I owe him a great deal.

Bruce was working on the characterization of the enzymes in the histidine

synthesis pathway in Salmonella. We had completed the work on three of the

enzymes when Bruce left for a month to work in Arthur Kornberg’s department in

St. Louis. As he went out the door, he handed me a tube containing the substrate for

the last enzyme in the pathway and asked me to characterize that enzyme as we had

the others. The only problem was that when I took the spectrum of the substrate,

I found that its synthesis had gone wrong. I had no substrate to work with.

Long-distance telephone calls, at that time, were very expensive. It was unthinkable

to try to call Bruce and ask for instructions. So I took the question to one

of the senior investigators in our department. “Find something useful to do. Bruce

will be home soon,” he responded.

I cogitated over this for a bit and then decided to apply some of the operon

thinking I learned in Paris to the histidine pathway. I tried out some conditions

I thought would reveal coordinated regulation of the expression of the enzymes

that were already characterized and, to my surprise, readily found such conditions.

Bruce was pleased with this finding when he returned, but he put it aside until the

entire pathway was properly characterized. After a couple of months, I left Bruce’s

lab because I was getting along in my second pregnancy. Bruce later completed

the operon study with Barbara Garry. He included me as an author on the paper,

which became my first peer-reviewed publication (1).

THE STANFORD YEARS

LEN: The opportunity to move to Stanford came as a complete surprise. Joshua

Lederberg visited Harry Eagle’s laboratory toward the end of my required Public

Health Service “hitch.” We had a long discussion about the future of mammalian

somatic cell genetics and the progress I had made thus far in developing useful

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markers for the cell lines I had chosen. I was a bit preoccupied at the time because

I was in the midst of negotiating a permanent position at the NIH. However, the

talk with Josh was really a delight.

Later in the week, Harry Eagle calledmeinto his office and suggested that I delay

a bit before making any commitments at the NIH. I didn’t make the connection

with Josh’s visit and was somewhat mystified. However, a few days later, a letter

arrived inviting me to consider a faculty position in the Genetics Department that

Josh was in the midst of establishing at Stanford!

Our parents considered the offer a disaster. California was still a long, long way

away from Brooklyn, and they now had two grandchildren they wanted to help

raise. Lee and I were also somewhat negative about returning to the West Coast.

However, just after the offer came, we made a trip to New York that reset our

direction.

We came to New York so I could attend the annual Federation of American

Scientists meeting. Martin Kamen also attended the meeting andwound upwalking

with me at its close from upper Manhattan to the Times Square subway station.

I had met Martin when I was at Caltech, at a benefit party Linus Pauling gave to

help him raise funds for his legal fees. He was fighting the INS decision to revoke

his passport (another McCarthy victim).We talked a bit about this, and then I told

him about the possibility of going to Stanford in Josh’s new department. By the

end of the walk, I realized that going to Stanford was a chance of a lifetime and

that there was no way I could turn down a position there, if I got it.

Why was Stanford so exciting? Well, with urging from Henry Kaplan, head

of Radiology at the Stanford Medical School and the pioneering developer of

treatment protocols for Hodgkins disease, Stanford President Wallace Sterling

had mustered the resources to upgrade the Medical School to a first-rate institution

with the twin goals of forefront research and excellent clinical practice. Arthur

Kornberg, who would win the Nobel Prize in 1959, was recruited as chairman

of Biochemistry, and he in turn recruited the cream of the department he chaired

in St. Louis. Joshua Lederberg, who couldn’t interview me until February (1959)

because he had a date with the Nobel Prize in December 1958, was brought in as

chairman of Genetics.

I was the first faculty member Josh recruited. Josh’s (first) wife Esther was

also in the department. One reason she and Josh had chosen Stanford was that,

unlike Berkeley and many other schools at the time, there were no nepotism rules

at Stanford that prevented her from working with Josh. I noted this, although Lee

planned to look for a job in another department or possibly go back to school to

get the degree(s) she wanted.

In September 1959, the Medical School began moving into its new building,

which housed both the basic sciences and the hospital. Lee and I (and our two

children) arrived just as this was happening. The Biochemistry Department was

already in its quarters. Space had been opened for Josh’s lab and the Genetics

office, but little else was ready. The landscaping had not even been started, so the

building stood in the middle of a hot, dusty field.

TWO LIVES INTERTWINED 9

I was given temporary laboratory space in the nearby Applied Physics building

and set up my lab so that I could get the cultures that I had shipped from the NIH

growing. But California weather was not kind. September that year turned out to

be mercilessly hot, and the building I was in didn’t have air conditioning. As it

turned out, it would have been better if my incubator had had a water cooled, rather

than a water heated, jacket! Fortunately, I was able to recover from frozen stocks

much of what I had lost.

Lee beganworking withmearound this time. I had already applied for and gotten

a grant to support my somatic cell genetics work. The funding was available, but

the delays in completion of the Genetics space put everything else into chaos. I

had made a list of equipment I wanted purchased before I arrived, but none of it

had been ordered. The Genetics office was overworked and understaffed, and my

cultures were cooking in the incubator. Lee decided to pitch in for a while to help

me get started. Best decision we ever made!

Stanfordwas great for another reason. During McCarthy times, the University of

California and many other schools required faculty to sign a loyalty oath swearing

that they were not now, and had never been, a member of the Communist Party

or any other organization that advocated the overthrow of the federal government.

Because the list of proscribed organizations was created at the whim of people

who rose to power ferreting out supposed communists, its sweep was extremely

broad. Many faculty members found the requirement of a loyalty oath repugnant

and refused to sign.

Stanford supported this view by refusing to institute a loyalty oath and by

hiring people who left other institutions rather than sign such an oath. A number

of eminent Berkeley physicists moved en masse from the Berkeley to the Stanford

Physics Department. We were pleased to have the opportunity to meet and work

with these physicists in the years that followed.

We Become Immunologists

Shortly before coming to Stanford, Josh Lederberg had spent some time in Australia

with Sir MacFarlane Burnet, who was head of the Hall Institute in Melbourne.

Josh and Sir Mac applied genetic thinking to the immune response and came up

with the idea that antibody responses reflect the clonal selection of cells that are

individually committed to producing antibodies that recognize, and are triggered

by, the immunizing antigen.

This so-called clonal selection theory predicted that individual cellswould make

antibodies specific for a single antigen and stood in opposition to instructive theories

that predicted much more plasticity for individual cells. The clonal selection

hypothesis ultimately won out. However, at the time Lee and I arrived at Stanford,

the jury was still out.

To do the studies that would test this hypothesis, Josh appointed two young

visiting faculty members: Gustav Nossal, who later followed Burnet as head of the

Hall Institute, and Olli Makela, who later returned to Finland to do immunology

10 HERZENBERG ¥ HERZENBERG

research and eventually became Dean of the Medical School in Helsinki. Gus

and Olli, and the mouse facility they would need, were to be housed in the same

corridor as the lab being completed for my use. So the move to the new building,

which occurred about December 1959, put my somatic cell genetics group right

next to one of the most exciting immunology projects of the time.

The First Immunology Studies

LEE: By the time we came to Stanford, Len had already developed an interest in

the practical side of immunology. Just before leaving the NIH, he arranged to visit

George Snell at Bar Harbor to discuss the idea of using mouse histocompatibility

antigens, rather than drug sensitivity, as somatic cell markers in cultured cell lines.

Len thought itwould be neat to use cytotoxic antibodies to the H-2 antigen (thought

to be a single entity at the time, now recognized as the MHC) to select cell surface

antigen variants in lymphocyte and other cell lines. However, hewondered whether

this would be practical. So he went to talk to George, who was very encouraging

and offered some antibodies for this purpose in case Len needed them.

This idea lay fallow until we sorted out all the problems involved in getting the

lab set up. However, once this was accomplished, Len suggested to me that I take

on the job of anti-H-2 antisera so that we would have our own reagents with which

to select variants. Of course, I had never touched a mouse and knew nothing about

how to proceed other than what I could read. Nevertheless, I took on the job. Len

and I both liked it as a project for me because I could work independently at my

own speed without creating for him a bottleneck on a critical path.

To figure out how to start, I went knocking on Gus and Olli’s door. They did

indeed know how to proceed, and they showed me how to take out spleens, use

spleen cells to immunize the mice, and do tail bleeds to collect the sera. They

weren’t much help in setting up the erythrocyte agglutination assays that were

used at the time to titer the sera. However, with their and Len’s advice and several

quite good papers on the subject, I managed to get a test going and learn to reliably

read it.

There was already a great deal of serologic evidence characterizing the genetically

distinct H-2 antigens expressed by various mouse strains. Because C57BL

mice were known to make strong antibodies to the DBA/2 H-2, and because both

kinds of mice were available from a local commercial breeder, I chose this combination.

In addition, because female C57BL mice that had been retired from the

breeding colony were large and could be obtained quite cheaply, I chose these

mice to immunize. I got very good responses and was able to collect lots of good

antisera that Len could use for selection.

Surprisingly, however, some of the control sera that I took from the breeders

before immunization turned out to have lowbut clearly positive levels of antibodies

that agglutinated DBA/2 rbc but were clearly negative against the serum producer

strain (C57BL). A bit of detective work soon showed that many of the retired

breeders that we had purchased had been out crossed to DBA/2 to make F1 animals

TWO LIVES INTERTWINED 11

that were in high demand. Thus, by following my nose, I had discovered a potential

model for human Rh immunization during pregnancy (2).

Some time later, I showed these data to Ray Owen, the Caltech professor who

taught the immunogenetics course I had taken. Ray shocked me by asking when

I was going to publish it. I stammered and stuttered a bit until Ray finally said,

“Well, if you are serious about being a scientist, then I guess you have to publish

this.” So I did. For this and many other reasons, I often refer to Ray as the closest

I ever had to a graduate professor.

Focus on H-2 Antigens

LEN: With a plentiful supply of anti-H-2 antisera, I decided to phase out my drugresistance

work and focus on using these sera for genetic studies with mouse cell

lines. First, however, I needed to do some characterization of the H-2 antigen, at

least to the point where I knew what it was. There was general confusion on this

at the time. Immunologic evidence had located H-2 on the cell surface of many

cell types. However, while some people thought the antigen was composed of

carbohydrate or protein, no lesser a light than Peter Medawar, who would later

be awarded the Nobel Prize for discovering adaptive immune tolerance, thought

that H-2 was made of DNA. We soon laid this issue to rest by isolating plasma

membranes and characterizing the H-2 antigen(s) associated with the membranes

as a protein or glycoprotein (3).

Working on H-2 drew me ever closer to the immunology community at Stanford.

Gus and Olli became close friends as well as wonderful colleagues who

loved discussing science as much as I did. Avrion Mitchison, who later headed

a productive Immunology Department at University College, London, was also

appointed as a visiting professor by Josh and began occupying the lab next door

within the year. Together, we established an immunology journal club, which met

one evening a week at my house as a no-holds-barred discussion in which we examined

methodology, evaluated experiment design, questioned conclusions, and

argued theory. The descendant of this journal club still functions in our laboratory

today, with much the same rules.

Gus and Olli were highly focused on testing the clonal selection theory (4)

during this time. Their approach was to isolate individual antibody-producing

cells and determine whether a single cell made antibodies to one or both of

a pair of immunizing antigens. Their data, although limited by the number of

cells they could isolate and test, clearly favored clonal selection. Mel Cohn,

in the Biochemistry Department three floors up, with colleagues Lennox and

Attardi at other institutions, were holding down the instructive corner of the

argument.

At the time all this was happening, I didn’t have a notion that I would one

day develop an instrument (the FACS) that would make it possible to resolve this

question. However, once we got the FACS running, we returned to these issues in

studies that became a major focus of our laboratory for several years.

12 HERZENBERG ¥ HERZENBERG

Enter Immunoglobulin Allotypes

In the spirit of the times, the clonal selection debate did not sour my relationship

with Mel Cohn, with whom it has always been fun to argue about anything. In

any event, when Mel decided to leave Stanford in 1962, he “willed” the medical

student working in his laboratory to me. John Wunderlich thus joined our group,

bringing with him a project focused on producing antisera that would distinguish

between antibody molecules produced in different mouse strains and putatively

encoded by different alleles in those strains (5).

Ultimately, this project blossomed into a full-scale study of the genetics of the

Ig heavy-chain (IgH) chromosome region (6). Long before the structure of the IgH

region was defined by molecular methods, studies with anti-isotype and antiallotype

antisera showed that IgH isotypes are encoded by a series of closely linked

loci and that various mouse strains have distinctive alleles at these loci. The IgH

isotypes were defined by other laboratories; we produced many of the antiallotype

sera and used these sera in genetic studies (gel immunoprecipitation and radioimmune

assay) to demonstrate the close linkage of several of the IgH (isotype) loci.

In addition, we defined a series of IgH haplotypes based on the combinations of

alleles represented at the IgH constant region loci on the IgH chromosome in each

of the standard mouse strains and showed that these were codominantly inherited.

Interestingly, the IgH haplotypes defined in this way provided the basis for the

Jan Klein and Don Shreffler model for organization of the MHC chromosome

region.

We reported our IgH genetic studies at a Cold Spring Harbor Symposium (7)

at which Henry Kunkel presented evidence for similar close linkage of the human

IgH loci. Together, these studies solidified a paradigm that was extended by the

recognition of additional loci and haplotypes and laid the groundwork for the modern

understanding of Ig rearrangement, isotype switching, and haplotype (allelic)

exclusion during B-cell development.

Regulation ofMemory-B-Cell Expression

LEE: Although I was working actively on allotype genetics, I maintained an independent

interest in maternal immunization to fetal antigens (H-2 in particular)

and in the effects of such immunization on the developing fetus. Therefore, when

maternal antiallotype antibodies were shown to pass to the fetus and to delay the

initial appearance of the paternal allotype in allotype heterozygotes, Len suggested

that I get this to work with some of our mouse strains. I did, further extending my

independent work in the lab.

Ultimately, Len’s suggestion led us to the discovery of “chronic” allotype

suppression. This occurs when SJL males are mated to immunized BALB/C females

producing high-titer antiallotype antibodies reactive with the paternal Igh-1b

(IgG2a) allotype. This finding then led to the discovery and characterization ofCD8

suppressor T cells that control the expression of IgG2a (Igh-1b) memory B cells

without impacting survival of the memory population (8).

TWO LIVES INTERTWINED 13

While exploring the mechanism(s) underlying allotype suppression, we recognized

(as others had before us) that priming with typical protein antigens enabled

a strong secondary response to the determinants present on the priming antigen.

Such priming also resulted in suppressed responses to new epitopes such as haptens

introduced on the priming antigen at the time of the secondary challenge.

Because this suppression persists when carrier/hapten-carrier-immunized animals

are challenged with the new epitope (hapten) on a different carrier protein, we

refer to it as epitope-specific suppression (9).

Although these studies were highly rigorous, they were not met with universal

acclaim, perhaps because of the confusion they introduced and perhaps because

they occurred just at the dawn of the molecular era in immunology. Nevertheless,

the findings are “alive and well” in the vaccine world, where they have been confirmed

with a variety of antigens and provide an important caveat when generating

vaccine strategies. Similarly, the immunoregulatory-circuits model we had constructed

just prior to beginning the epitope-specific work (10), which predicted

much of what we found, was not roundly embraced by immunologists, but it too

is alive and well, I am told, among today’s immune-system model builders.

THE FACS

LEN: As I became more deeply involved in immunology, I became increasingly

aware of the need to characterize and isolate the different kinds of lymphocytes

that were beginning to be visualized with fluorescent-labeled antibodies under the

microscope and studied functionally by sensitivity to complement-mediated depletion

after treatment with antibodies (in conventional antisera). The need for better

cell-isolation methods here dovetailed completely with the need for developing a

method for positive selection of variants in the somatic-cell genetics projects that I

was also engaged in. So I started asking around to see whether anyone had solved

this problem.

I soon found out that a group at Los Alamos (led by Mack Fulwyler and Marvin

Van Dilla) had developed a machine that could examine and sort large numbers of

cell-sized particles on the basis of particle volume. I immediately planned a trip

to see whether I could convince them to add a fluorescence-detection system so

I could use their machine to measure the amount of fluorescence associated with

individual cells and to sort cells according to this measure in addition to volume.

They demurred, saying that this “was not part of their mission.” [They were funded

to build a machine to count and size particles, not cells, obtained from the lungs of

mice and rats sent up in balloons to inhale debris generated by atomic-bomb testing

(11).] I persisted, and they finally agreed to give me a set of engineering drawings

and the permission to use them as the basis of a machine designed to distinguish

cells labeled with fluorescent antibodies. Little did I know when I brought these

plans back to Stanford that I was starting on a lifework that continues today as a

major activity in our laboratory.

14 HERZENBERG ¥ HERZENBERG

Back at Stanford, I took advantage of my close proximity to the instrumentation

research laboratory set up by Joshua Lederberg to look for life in outer space (on

Mars or on the Moon). I asked the engineer I knew best to look at the plans and

estimate the cost to replicate the Los Alamos machine. He came to me a few days

later and said, “Okay, I’ve got good news and I’ve got bad news. Which do you

want to hear first?” I opted for the good news, and he said, “Well, I think the

machine can be built here and I’ve completed a list of parts to be ordered.” I asked

what it would cost, and he answered, “Something like $14,000.” That was a lot

for those days, but it could probably be managed. So I asked him, “What’s the bad

news?” He answered, “The bad news is that I’m leaving Stanford. I’ve got another

position.”

I next talked to Josh and the head of the Instrumentation Laboratory. They

agreed that despite the loss of this key engineer, they could provide the engineering

help I needed for the project. I went to Henry Kaplan, who was head of the

Radiology Department and was working on thymic function and development

(Irving Weissman worked with him). I told him how I thought a fluorescencebased

cell-analysis and -sorting machine could be used to study the thymus and

asked him to join me in funding the development of this machine. He agreed. I put

up $7,000 from my somatic-cell genetics grant, he put up the remaining $7,000

needed to meet the estimate, and the project got under way.

I didn’t do any of the engineering on this project.However, Iwas deeply involved

in the daily development. I was essentially the head of the design team and took

responsibility for assuring that the machine would be usable by scientists doing

immunological or genetic studies. For example, at one meeting, the engineers told

me that the best they could do was to take data from about one million cells in

an hour. This was too few to be useful, so I insisted that they either increase the

speed by an order of magnitude or close the project down. At first there was some

discussion about “repealing the laws of physics,” but eventually an engineer came

up with a solution and we were off and running again.

I was also responsible for getting new capabilities designed and tested. I loved

this role because it encouraged me to think broadly about potential applications for

the nascent FACS and to develop collaborations within and outside our laboratory

to generate and test these kinds of ideas. In fact, although FACS development has

long since ceased to be an activity occurring solely within my purview, I still enjoy

the development of new FACS applications and the scientific breakthroughs such

development engenders.

Our first cell-sorting paper was published in Science in 1969 and was entitled

“Cell sorting: automated separation of mammalian [plasma] cells as a function of

intracellular fluorescence” (12). The instrument we used for this study had a xenon

light source, which we replaced with a laser shortly thereafter.

By 1972, we had developed a much improved instrument and decided to call

it the Fluorescence-Activated Cell Sorter (FACS). The engineering team was also

much improved because I was able to recruit Richard Sweet, inventor of the ink-jet

printer, to head the team. In essence, I pointed out to Dick that the sorting module

TWO LIVES INTERTWINED 15

in the FACS was based on his invention and asked him to join our group. He

responded, “There’s nothing I’d like to do more. I’d like to see biological applications

of my inventions.” And see he did, as he applied himself to the development

of several of the core features still with us in the modern FACS instrument.

Dick’s initial work generated a paper, published in the Review of Scientific

Instruments, that was really the first one describing the modern FACS (13). He also

joined us as an author of a 1976 Scientific American article in which we introduced

the FACS and the idea of using this novel instrument to track the expression of

genes encoding surface molecules that distinguish various kinds of lymphocytes

and other cells (14).

FACS Goes Commercial

The next major milestone in the development of the FACS was a meeting I had

with a vice president of Becton-Dickinson (BD), parent company of the current

BD Biosystems (BDB), and with Bernie Shoor, then a local BD representative who

ran an engineering group in the Stanford area. Bernie and the vice president came

to me because they wanted help with making (conventional) antibodies. I changed

the subject and said, “Well if you’re interested in making antibodies, then you’re

interested in immunology. The most exciting thing in immunology right now is

our fluorescence-activated cell analysis and sorting [FACS] instrument, which we

have been developing for some time and is now working!”

Bernie was interested in the machine but didn’t think it was commercially

viable. “I think maybe we could sell 10 of these instruments worldwide,” he said.

I thought 30, or possibly as high as 100 sales were more likely, but neither number

seemed high enough to BD to support turning FACS into a commercial machine.

BD, in the person of Bernie Shoor, would have walked away from the venture if I

hadn’t gotten an NIH contract that would let me subcontract the building of two

such instruments to Bernie’s group and let me collaborate in the effort. It’s also

true, though, that I might not have gotten the contract if I did not have BD on board

to build it.

We eventually built two FACS instruments, one for Stanford and one for the

National Cancer Institute (NCI), which put up the money as part of the “war on

cancer” (15). Within a short time (as such projects go), we had a commercial

instrument to replace “Whizzer,” the breadboard model we had been running until

then. FACS-1 was later upgraded to FACS-2, which ran for many years both in

our laboratory and at the NCI. Ultimately, the NCI instrument wound up in an

NIH museum. Our instrument went to the Smithsonian Institute in Washington,

DC, and is presently on display at the Walter Reed Army Institute of Pathology.

The Walter Reed exhibit includes a tape recording of me describing some of the

early work our group did and some of the work done by Bernie Shoor and his

group.

After the NIH contractworkwas completed,BDsuccessfully marketed FACS-2

and we continued our independent development effort.Within the next few years,

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we had made some key improvements, including the addition of fluorescencecompensation

circuitry to correct for spectral overlap between dyes and fourdecade

logarithmic amplifiers to allow the full range of FACS data to be displayed

on a single data plot. In addition, we introduced the use of computers for data

collection and built the first software for FACS data computation and display.

Monoclonal Antibodies as FACS Reagents

LEE: In 1975, just about the time that Cesar Milstein and GeorgeKohler succeeded

in immortalizing spleen cells that produce antibodies by fusing them with a longestablished

myeloma cell line, Len arranged a sabbatical in Cesar’s laboratory at

the Medical Research Council (MRC) in Cambridge, England. He chose the MRC

to learn the new molecular biology methods (which he did). However, by the time

we reached Cambridge in the fall of 1976, the fusion work was in full swing,

and Len was quick to realize that the ability to produce monoclonal antibodies to

cell-surface determinants would remove what had come to be the most irritating

restriction to FACS work at the time.

The conventional antibody reagents that we were using for FACS studies were

made primarily in mice or rats and were always in short supply. Furthermore, the

specificitywas always questionable because the animals were immunized with cell

preparations that contained many different potential antigens. Finally, the ability

to produce directly conjugated reagents was very limited, making background

staining by the second-step reagents a major problem. No wonder then that Len

was anxious to tap this new monoclonal reagent resource.

Cesar, on the other hand, had not attended the many meetings we had at which

discussions of potential problems with conventional antibody reagents had been

narrowed down to the need for groups to exchange staining reagents before the

findings could be evaluated. Therefore, Cesar was not highly motivated to have

Len delve into the monoclonal technology and urged him instead to pursue the

molecular biology studies he had come to do.

The solution to this came when Vernon Oi, then a graduate student in our

laboratory at Stanford, came to Cambridge for a prolonged stay. I was working,

in principle, at the Babraham laboratories with Arnold Feinstein. However, I had

not made much use of the space Arnold gave me because I had to write several

chapters for the Weir Handbook of Experimental Immunology as well as several

papers that had piled up before I left Stanford. Arnold was pleased to let Vernon

take my place, and with agreement from Cesar, he outfitted a laboratory in which

Vernon (with help from Len and me) could make a set of monoclonal antibody

reagents that would detect allotypic determinants on IgG molecules (16).

We brought this technology home at the end of the sabbatical year and, with

a FACS available for screening for antibodies to cell surface determinants, began

making a series of monoclonal reagents (17) to mouse MHC and other cell-surface

molecules (16, 18). Shortly thereafter, we made a unilateral decision to make our

monoclonal reagents, and the cell lines that produced them, freely available to the

TWO LIVES INTERTWINED 17

scientific community. I had the pleasure of announcing this at a large MHC workshop

meeting and was pleased when Baruch Benacerraf approached me after the

session to compliment our laboratory specifically on this decision. Breaking into

the circle surrounding me, he said, “I would like to shake your hand.” And he did!

Len also recognized at this time that distributing cell lines that produce important

monoclonal reagents would not be sufficient to ensure the availability of these

reagents to the overall immunology community. While still in Cambridge, he had

phoned Bernie Shoor to suggest that he get BD to set up a commercial mechanism

for producing and distributing monoclonal FACS reagents. It took some time for

this to occur, but BD ultimately set up a business whose growth and importance

to research and medical practice has well validated the original idea.

Interestingly, neither BD nor we thought it necessary or appropriate to patent

the monoclonal reagents that the BD monoclonal center was producing, or even

to restrict the dissemination of the cell lines that produce these antibodies. Bernie

felt, and was proven correct, that people would prefer to buy well-characterized

fluorochrome-conjugated reagents rather than produce these reagents themselves.

This view is probably more correct now than it was at the time. However, it became

untenable as patents for biological material became commonplace and suits for

patent infringement began invading the biomedical arena.

Some time later (1982), Vernon Oi and I teamed up with Sherie Morrison (on

sabbatical leave with Paul Berg at Stanford at the time and now at UCLA) to make

human/mouse chimeric antibodies in which the antibody specificity was encoded

by variable-region genes derived from mouse and the heavy-chain constant region

was encoded by human IgH genes (19). Because we believed that chimeric antibodies

of this type were likely to be useful as functional antibodies and therapeutic

reagents, we applied for a patent for this molecular method (issued in 1998). We

have been pleased to see the method applied by others, e.g., in the production of

chimeric anti-TNF-® used in the treatment of human autoimmune diseases.

FACS: The First Biotech Instrument?

If the biotechnology (biotech) industry can appropriately be characterized as an

industry built around defining, measuring, and making use of gene expression in

biology and medicine, then the FACS as we built, described, and used it in the

early 1970s readily qualifies as a biotech instrument. In fact, to our knowledge,

it is the first such instrument. In addition, Garry Nolan (Medical Microbiology

and Immunology, Stanford) points out that FACS should be recognized as a key

proteomics instrument, since it has been used in numerous studies to define the

functions and demonstrate the interactions of surface and intracellular proteins.

Although titles shouldn’t really matter in science, it is appropriate to grant the

FACS these distinctions. Similarly, it is appropriate to congratulate Bernie Shoor

and BD for having had the foresight to build the first biotech company and to lay

the foundations for it to grow to its current status as Becton-Dickinson Biosciences

(BDB).

18 HERZENBERG ¥ HERZENBERG

Len, of course, has been honored many times for his innovative role in developing

the FACS and demonstrating its applications in biology and medicine. Notably,

he was cited for this work when elected to the National Academy of Sciences in

1982.

Breaking the FACS Color Barrier

LEN: At the beginning of the 1980s, we realized that the immunology and other

studies that we wanted to do were limited by the number of individual fluorescence

measurements (sometimes called parameters) that a single-laser instrument

could make on individual cells. There were enough markers known on T cells, for

example, to suggest that multiple subsets existed. However, we recognized that

using these markers effectively requires their simultaneous measurement on individual

cells. Measuring their expression two by two, or even three by three, is not

adequate. Information is lost when the measurements are separated and cannot be

regained by trying to merge them during analysis. David Parks, then a member and

later the leader of our FACS Development Group, solved this problem by extending

the FACS-2 to create a dual-laser FACS instrument that would, at a minimum,

double the number of markers we could measure on a given cell. In addition, he

independently developed single-cell cloning and added this capability to the dual

laser instrument (20–22).

This was not the first dual-laser FACS (one had already been created by an

instrumentation research group in Germany). However, it was the first dual-laser

instrument put into routine use for immunologic studies and hence was the first instrument

to demonstrate the effectiveness of using multiparameter FACS methods

for distinguishing lymphocyte subsets and for sorting these subsets for functional

studies. Many of the key findings made over the years by our group and by other

research groups at Stanford were enabled by the development of this dual-laser

instrument and its installation in the Stanford Shared FACS Facility, which Len

helped to organize some years ago and which David Parks now directs.

We put the dual-laser instrument into routine service in 1983. Roughly 15 years

later (1998), we put into operation a hybrid instrument (BD bench, Cytomation

electronics) that provides three independent laser illuminations and can simultaneously

measure up to 11 distinct fluorescence emissions from individual cells.

The number of markers measured with this high-definition (Hi-D) FACS (23–25)

instrument, and with our recently purchased BDB Hi-D instruments (FACS DIVA

and ARIA), has grown from an initial 8 to the current 11, nowthe standard for most

work in our laboratory. Mario Roederer and his group at the NIH have extended

FACS DIVA and located additional fluorescent dyes that can be measured simultaneously

to further increase the number of measurements that can be made per cell.

The Soft Side of FACS

LEE: The original FACS data were collected by photographing histograms traced

on an oscilloscope screen. Although these were the early days for using computers

TWO LIVES INTERTWINED 19

to collect data from laboratory instruments, we once again were able to capitalize

on our connection with Josh Lederberg’s exobiology engineering group and,

with their help, began using the Digital PDP-8 computer to collect FACS data.

Wayne Moore, who joined the FACS development group shortly thereafter and

has since built or supervised the building of all of our FACS software, moved the

FACS data collection and analysis to the PDP-11 platform. On this platform, he

developed models for much of the data analysis and display methods that are still

in use, including the equal-density (probability) contouring method that is today’s

standard.

FACS/Desk, which Moore introduced at about the same time that our duallaser

FACS was put into operation (26), was built on a VAX-11/780 platform and

offered a nonprocedural (keystroke rather than command line) user interface. This

interface, which had windows that opened and asked for user input, foreshadowed

what I was later to see in the Apple Macintosh windowing environment.

Sometime around 1980, Len had to raise nearly half a million dollars to buy

the VAX computer and build the specialized computer facility necessary to house

it, but we have always considered this well worth the trouble. The capabilities that

Moore’s full FACS/Desk system provided, and still provides, have enabled countless

large multiparameter experiments and have provided a permanent, searchable

record of all FACS experiments done in the Stanford Shared FACS Facility.

For the past several years, we have been working on a replacement for FACS/

Desk. Some time ago, we did the initial designs for a new FACS analysis package.

These provided the basis for Mario Roederer’s extensions and ultimately for the

commercially developed FlowJo package (TreeStar.com), which is widely used

today. At present, we are migrating data stored in FACS/Desk to our new FACS

DataStore, whose capabilities are much improved over the older system, and have

completed an initial version of a searchable Directory Server that can be closely

integrated with the new DataStore.

We, in collaboration with Mark Musen, Medical Information Sciences, Stanford,

and Stephen Meehan, Meehan Metaspace, are also about to complete the

first version of a FACS protocol editor (FacsXpert) that provides an advanced user

interface coupled to knowledge-based technology to facilitate design of 12-color

FACS staining protocols. FacsXpert is also designed to “painlessly” capture the information

(metadata) necessary to annotate data for analysis output (e.g., for axes

and table heads) and to facilitate searches with the Directory Server. Ultimately,

we hope to make all these capabilities available to the scientific community and

to extend the system to take and store data from multiple instruments. This goal,

however, may have to wait until we can find a willing and appropriate commercial

partner.

Len has been both contributive and supportive in this software development

effort. However, in some ways it has been very much “my baby.” Although I have

written only a small part of the overall system (specifically, the FACS Facility instrument

scheduler), I have frequently participated in the design process and have

opened relevant collaborations with colleagues in the Stanford Computer Science

20 HERZENBERG ¥ HERZENBERG

and Statistics Departments. In addition, I pioneered the connection of FACS/Desk

and FACS analysis output to the SAS Institute JMP statistics package to enable

analysis of data from our HIV clinical trials. For this effort, and for the development

of the overall FACS/Desk system, we were awarded the Computer World

Smithsonian Award in recognition of our visionary use of information technology

in the field of medicine.

A Note about Innovation

Although all the key flow cytometry technologies that we developed were eventually

adopted as standards by the commercial and academic flow-cytometry community,

we have routinely encountered substantial resistance to the initial spread of

these technologies. Biological innovations such as the use of monoclonal antibodies

as FACS reagents were readily and rapidly accepted (27). However, technological

innovations not part of the biological idiom fared less well, particularly when

statistical or mathematical treatments were involved. Hopefully, this will change

as these modes of data analysis become more common within the biomedical

research community.

Continue to Read, In His Own Words

THE FACS IN THE SERVICE OF IMMUNOLOGY (AND VICE VERSA)

Profile depicted in NYT article by Douglas Martin on 11/10/2013:

Leonard Herzenberg, Immunologist Who Revolutionized Research, Dies at 81

By DOUGLAS MARTIN

Published: November 10, 2013

http://www.nytimes.com/2013/11/11/us/leonard-herzenberg-immunologist-who-revolutionized-research-dies-at-81.html?emc=eta1

Read Full Post »